Melanocyte Damage Research Articles

IgG Anti-Melanocyte Antibodies Purified from Patients with Active Vitiligo Induce HLA-DR and Intercellular Adhesion Molecule-1 Expression and an Increase in Interleukin-8 Release by Melanocytes

An immunologic hypothesis is currently proposed as a possible pathogenesis of nonsegmental-type vitiligo. IgG antibodies against melanocyte surface antigens exist in the serum of patients with vitiligo vulgaris. IgG anti-melanocyte antibodies were reported to induce melanocyte damage in vitro by a complement-mediated mechanism and antibody-dependent cellular cytotoxicity. Perilesional melanocytes express major histocompatibility complex class II antigens and a higher intercellular adhesion molecule-1 compared with those in normal skin. The purpose of this study was to determine the role of IgG anti-melanocyte antibodies in the inappropriate expression of major histocompatibility complex class II antigens and intercellular adhesion molecule-1 on melanocytes. IgG anti-melanocyte antibody samples were purified from the individual serum of patients with active vitiligo. After incubation of IgG anti-melanocyte antibodies with cultured melanocytes, the results revealed: (i) IgG anti-melanocyte antibody stimulated HLA-DR expression on melanocytes; (ii) intercellular adhesion molecule-1 expression on melanocytes was significantly induced by IgG anti-melanocyte antibodies; and (iii) IgG anti-melanocyte antibodies induced an increase in interleukin-8 release from melanocytes. The major histocompatibility complex class II molecules expressed in melanocytes can present antigens to CD4 helper cells as antigen-presenting cells and elicit an immune response. Intercellular adhesion molecule-1 is an important adhesion molecule involved in leukocyte and parenchymal cell interaction and thus plays an essential part in immunologic and inflammatory reactions. It is reasonable to speculate that abnormal expressions of HLA-DR and intercellular adhesion molecule-1 on melanocytes by IgG anti-melanocyte antibodies would present vitiligo antigens and allow the antigen-specific immune effector cell attack that results in melanocytotoxicity.

Clinical features of Ota's naevus in Koreans and its treatment with Q-switched alexandrite laser.

Ota's naevus is a fairly common pigmentary disorder in Asians. Recently, encouraging results in the treatment of Ota's naevus have been obtained, but most of these concerned the white skins of Caucasian patients. Our purpose was to examine the clinical features of Ota's naevus in Koreans and to assess the clinical outcomes and histological changes induced by a Q-switched alexandrite laser at 755 nm. Eighty-seven Koreans with Ota's naevus were studied; the peak age of onset was during the first decade and adolescence. The infraorbital area was the most frequent site and black or dark brown colours predominated. Improvements were achieved in 52 patients (77%). Better results were obtained in unilateral lesions and patients who received a greater number of treatments. Mild hyperpigmentation after treatment was noticed in 14 patients and mild hypopigmentation in eight patients. However, all of these were reversed in time. Hypertrophic scarring or secondary infection did not occur. The histology of laser-irradiated lesions showed selective thermal damage of melanocytes in the upper dermis and the elimination of upper dermal pigmentation. Our clinical data demonstrate the usefulness of the Q-switched alexandrite laser for the treatment of Ota's naevus in brown skin.

G2 accumulation and melanin overproduction in malignant melanocytes treated with a new nitrosourea.

Cystemustine (N'-(2-chloroethyl)-N-(2-(methylsulphonyl)ethyl)-N'-nitrosourea), a new anticancer chloroethylnitrosourea (CENU) is being tested in a phase II clinical trial of disseminated melanoma. The antitumour effect of this drug is mainly due to DNA damage in malignant melanocytes. Recently, we have shown that this damage can induce apoptosis in some melanoma cell lines. In others, apoptosis is not clearly observed, although there is a strong cytostatic effect. In this paper, we have characterized the cytological effect of cystemustine on murine malignant melanocytes (B16 cell line) which are resistant to apoptosis induced by this CENU. The results show that 3 days after cystemustine treatment, these melanocytes had accumulated in phase G2 of the cell cycle. There was then a strong morphological modification during a long cytostatic phase up to 30 days after treatment. During this cytostatic phase, there was uncontrolled DNA synthesis and marked swelling. Also, tyrosinase activity, melanin content and the number of mature melanosomes were greatly increased. These results suggest that when malignant melanocytes are not able to undergo apoptosis after treatment with CENU, they accumulate in G2 and this is followed by enhancement of melanogenesis.

UVA-induced oxidative DNA damage in human melanocytes is related to their (pheo)melanin content

UVA-induced oxidative DNA damage in human melanocytes is related to their (pheo)melanin content

Increased Sensitivity to Peroxidative Agents as a Possible Pathogenic Factor of Melanocyte Damage in Vitiligo

To examine the sensitivity of vitiligo melanocytes to external oxidative stress, we studied enzymatic and non-enzymatic anti-oxidants in cultured melanocytes of normal subjects (n = 20) and melanocytes from apparently normal skin of vitiligo patients (n = 10). The activity of superoxide dismutase and catalase and the intracellular concentrations of vitamin E and ubiquinone were evaluated in cultures at the fourth or fifth passage. In addition, cells were exposed to various concentrations of a peroxidizing agent, cumene hydroperoxide (CUH, 0.66-20 microM), for 1 and 24 h. Compared to normal melanocytes, vitiligo melanocytes showed normal superoxide dismutase and significantly lower catalase activities and higher vitamin E and lower ubiquinone levels. At the concentration used, CUH did not significantly affect cell number or viability of melanocytes after either period of culture. On the contrary, vitiligo melanocytes were susceptible to the toxic effect of CUH after 24 h of continuous treatment at concentrations greater than 6.6 microM. The degree of CUH toxicity correlated strictly with the anti-oxidant pattern, defined as the ratio between vitamin E concentration and catalase activity, suggesting that the alteration in the antioxidants was the basis for sensitivity to the external oxidative stress. Our results demonstrate the presence of an imbalance in the anti-oxidant system in vitiligo melanocytes and provide further support for a free radical-mediated damage as an initial pathogenic event in melanocyte degeneration in vitiligo.

Alterations in IL-6, IL-8, GM-CSF. TNF-α, and IFN-γ Release by Peripheral Mononuclear Cells in Patients with Active Vitiligo

The purpose of this study was to clarify the relationship between the cellular and humoral immune components in the pathogenesis of vitiligo vulgaris. By using cytokines as indicators of peripheral mononuclear cell (MNC) function, we compared the effects of phytohemagglutinin (PHA) and purified IgG on MNCs derived from patients suffering from active vitiligo with those from normal controls. The results revealed (i) a significant increase in spontaneous production of IL-6 and IL-8 in patients; (ii) PHA, purified IgG from patients (IgG-anti-MC), or IgG from normal controls (N-IgG) induced a significant increase in IL-6 but diminished GM-CSF, TNF-alpha, and IFN-gamma release in patients; and (iii) IgG-anti-MC brought about a significantly higher stimulatory effect on IL-1beta and IFN-gamma production than N-IgG in normal controls. Immunologically, IL-6 can enhance melanocyte ICAM-1 expression, which may increase leukocyte-melanocyte attachment and cause melanocyte damage in vitiligo. A decrease in GM-CSF (an intrinsic growth factor for melanocyte) production may retard recovery from vitiligo by checking the proliferation of surviving melanocytes. A significant decrease in TNF-alpha and IFN-gamma production may partially explain the reduced inflammatory reaction in vitiliginous lesions. That IgG-anti-MC stimulates an increase in IL-1beta and IFN-gamma production in controls suggests that IgG-anti-MC may play a role in melanocyte destruction mediated by monocytes.

Lipoperoxidase activity of Pityrosporum: characterisation of by-products and possible rôle in pityriasis versicolor.

Modification of pigmentation and damage of melanocytes are characteristic features of skin colonisation of Pityrosporum orbiculare hyphae in pityriasis versicolor (PV). The yeast is lipophylic and lipid-dependent, capable of oxidising unsaturated lipid components of skin surface, i.e., unsaturated fatty acids, cholesterol and squalene (SQ). The oxidation of unsaturated fatty acids gives rise to dicarboxylic acids (DA) which behave, in vitro, as competitive inhibitors of tyrosinase. In this work, we further investigate the oxidase activity of Pityrosporum in vitro, by evaluating (a) the generation of lipoperoxides in cultures supplemented with fatty acids at various degrees of unsaturation; (b) the mechanism of SQ oxidation; (c) the chemical characteristics of some by-products of lipoperoxidation; (d) the formation of peroxisomes in fungal cells. In cultures supplemented with the saturated palmitic acid (C16:0) and monounsaturated oleic acid (C18:1 n-9), low amounts of lipoperoxides were detected by a spectrophotometric test, whereas in cultures supplemented with di-unsaturated linoleic acid (C18:2 n-6), significant concentrations were found. Gas chromatography-mass spectrometry analyses showed the generation of linoleic acid hydroperoxides both in Pityrosporum cultures and following incubation of acetone powder of the fungus with the unsaturated fatty acid, indicating the presence of a lipoxygenase activity in the fungus. In cultures supplemented with linoleic acid plus SQ, and increase of lipoperoxide generation was observed and trans-trans farnesal and squalene epoxides have been identified. Electron microscopic examinations have evidenced peroxisomes in cells grown in the presence of linoleic acid, whereas they were not detected in cultures supplemented with oleic acid and palmitic acid. The metabolic activities of peroxisomes, through the formation of hydrogen peroxide and the subsequent generation of hydroxyl radicals, may account for the peroxidation of SQ, which is not a substrate of lipoxygenase. Following these results, we propose a mechanism for DA generation by Pityrosporum metabolism and hypothesize that the lipoperoxidation process induced by lipoxygenase activity of the fungus may be the key to understanding the clinical appearance of skin manifestation of PV.

The expression of thec-kitreceptor by epidermal melanocytes may be reduced in vitiligo

The proto-oncogene c-kit encodes the transmembrane tyrosine kinase receptor that has a role in the growth regulation of various cell types including melanocytes. In the present study we have examined the expression of the c-kit protein in the skin of seven patients with vitiligo. Melanocytes positive for c-kit protein were observed in the basal layer in non-lesional skin and the mean number of 25.8 +/- 5.2 (per 200 basal cells) compared with that of 21.8 +/- 3.5 from six control subjects. In perilesional skin there was a reduction in the numbers of c-kit positive melanocytes (6.7 +/- 2.6) and this was especially noticeable in six of the seven patients. Such a reduction was less obvious following staining with MEL-5 and in only two subjects were the numbers of melanocytes below the normal range. This suggests that the reduction in c-kit staining was the result of decreased expression of the protein rather than a loss of melanocytes. No melanocytes, positive for c-kit protein, or after staining with MEL-5, were identified in lesional skin although isolated tyrosinase-positive melanocytes were seen in one subject. There was no apparent change in the numbers of mast cells expressing c-kit protein and the intensity of staining in the dermis even in lesional skin was similar to that in the controls. These results demonstrate that c-kit protein is present on melanocytes in adult human skin and that in perilesional skin of some vitiligo patients there is a reduction in the numbers of melanocytes expressing this receptor. Whether this may contribute to the defective melanocyte growth and/or survival that occurs in vitiligo or whether it is a consequence of melanocyte damage remains to be seen.

Comparison of Melanocytes and Keratinocytes in Ultraviolet-Induced DNA Damage per Minimum Erythema Dose Sunlight: Applicability of Ultraviolet Action Spectra for Risk Estimates

Action spectra are being used in risk estimates for ultraviolet (UV) damage. The purpose of our investigation was to compare the susceptibility of cultured melanocytes and keratinocytes to UV-induced DNA damage per minimum erythema dose (MED) and to determine whether the predictions made with action spectra agree with the damage actually induced. Genetic damage was measured as the number of T4-endonuclease V-sensitive sites (ESS). Predictions made with the action spectrum for the induction of DNA damage in melanocytes after irradiation with sunlight and a solar simulator were 15.9 and 13.2 ESS per 10(8) daltons per MED, respectively; with the action spectrum for the induction of DNA damage in keratinocytes the predictions were 12.1 and 9.8 ESS per 10(8) daltons per MED, respectively. To determine the actual damage per MED, cultured cells were irradiated with sunlight or a solar simulator, and MED was determined with an erythema UV meter. The induction of DNA damage in melanocytes after sunlight and solar simulator irradiation was 8.01 and 6.7 ESS per 10(8) daltons per MED, respectively, and in keratinocytes 7.49 and 7.12 ESS per 10(8) daltons per MED, respectively. This was considered to be in agreement with the predicted data. The use of action spectra for risk estimates in melanocytes appears justified.

Antioxidant status in the blood of patients with active vitiligo.

We have previously reported that patients with active vitiligo (AVP) have elevated urinary levels of catecholamine metabolites, such as homovanillic and vanilmandelic acids, irrespective of the form of the disease (acrofacial, segmental, generalized). We have suggested that abnormal release of catecholamines from autonomic nerve endings might play an etiological role in the onset and development of vitiligo through an overproduction of toxic (oxy)radicals in the microenvironment of melanocytes in the affected areas. In the present study we have investigated whether this suggested increase in radicals might be associated with an oxidative stress in the blood of AVP. We have analyzed by gas-chromatography mass-spectrometry, by high pressure liquid chromatography, by spectrophotometry plasma levels of vitamin E (Vit E), lipoperoxides (LIP), and polyunsaturated fatty acids of phospholipids (PL-FA), erythrocyte reduced glutathione (GSH), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) activities in 62 patients affected with different forms of active vitiligo (acrofacial, segmental, generalized) and in 60 age-matched controls. Our results show that blood levels of Vit E, SOD, GSH, GSH-Px activity, LIP and PL-FA in AVP were not significantly different from those of healthy age matched controls, indicating that melanocyte damage in vitiligo is not linked with a generalized oxidative stress.

In vivo depigmentation by hydroxybenzene derivatives.

Certain mono- and dihydroxybenzene derivatives are selectively cytotoxic for melanocytes in vivo, and can cause depigmentation of skin and hair. We produced selective melanocytotoxicity/hair depigmentation in C57Bl mice by injection of 0.032-1.0% p-t-butylcatechol (tBC) or p-hydroxyanisole (MMEH) in physiological saline. No depigmentation occurred on injection of 3,4-dihydroxyphenylalanine (DOPA) or 3,4-dihydroxyphenylacetic acid (DOPAC). Light- and electron-microscopic examination of biopsy specimens taken from depigmented areas indicates selective melanocyte damage as early as 2 h post-injection. Melanocytes from anagen hair are most susceptible to depigmentation. All four compounds are substrates for tyrosinase, but only tBC and MMEH generate their respective isolable 1,2-benzoquinones, tBCQ and MMEHQ. These caused depigmentation in C57Bl mice to a comparable degree to the parent compounds. DOPA- and DOPAC-quinones (DOPAQ and DOPACQ) are not spectroscopically detectable in solution, suggesting extremely low steady-state levels of these compounds. The net observed rate of reaction of the respective 1,2-quinone with 300 microM bovine serum albumin (BSA) in vitro varies widely, with tBCQ >> MMEHQ = DOPACQ >> DOPAQ. The results are consistent with a mechanism involving attack of -SH on melanosomal proteins and/or enzymes by tyrosinase-generated 1,2-quinones. This mechanism evidently differs from that involved in in vitro hydroxybenzene melanocytotoxicity of melanoma cells, in which active oxygen intermediates generated by hydroxybenzene autoxidation play a significant role. The most reliable prognosticator of in vivo depigmentation appears to be the ability of the depigmenter to form a spectroscopically stable 1,2-quinone which is capable of reacting with protein -SH.

An immunohistological study of cutaneous lymphocytes in vitiligo

In a study of 49 biopsies from the margins of depigmented cutaneous lesions in 18 patients with vitiligo, highly significant overall increases were found in CD3+, CD4+, and CD8+ T cells, though cell numbers in individual cases were often within the normal range. Many of the T cells were activated (MHC class II+, interferon gamma+), of CD45RO (UCHL1+) memory subset, and many expressed the cutaneous lymphocyte-associated antigen (HECA-452+) typical of skin-homing T cells. Immunohistologically, the most intense epidermal T-cell infiltration was present within 0.6 mm of the edge of the lesion in 10 of 13 double-stained sections with a clearly defined zone of melanocyte depletion. In 40 lesions from 17 patients seen 11-64 weeks after biopsy, no apparent association was found between T-cell numbers and disease activity as assessed by Köbnerization of biopsy wounds or spread of depigmentation. These findings are consistent with the hypothesis that lesional T cells rather than circulating antimelanocytic antibody may be responsible for the supposedly autoimmune but characteristically patchy destruction of cutaneous melanocytes in vitiligo. Nevertheless, many of the infiltrating T cells are probably innocent bystanders attracted by upregulated cell adhesion molecules near sites of melanocyte damage.

Ultrastructure of Melanocytes in Chronically Sun-Exposed Skin of Elderly Subjects

Chronically sun-exposed facial skin of three females aged 68, 71, and 78 years, and of a male aged 78, was examined by electron microscopy in order to study the condition of the epidermal melanocytes. Considerable heterogeneity of morphological and functional characteristics of the cells was observed. The majority of melanocytes were large, active, with occasionally lobulated or double nuclei, an appearance indicative of hyperstimulation. Some cells exhibited an appearance of having reached the end of an active life cycle and were labelled "aged." Others, in the upper end of the outer root sheath of hair follicles and adjacent interfollicular epidermis, presented a typically inactive appearance, indistinguishable from that of fetal melanocytes, or of those in unexposed skin of younger subjects. A cell with indented nucleus, fully melanised melanosomes, and hypertrophic Golgi apparatus was sporadically seen. Minute foci of dissociation of keratocytes were present, and melanocytes included in these were frequently disrupted. Swelling of mitochondria and cytoplasmic lipid droplets occurred sporadically within all the above variants of melanocytes. It proved difficult to establish criteria of specific sun damage of melanocytes. It is suggested that either the melanocytes exhibiting stimulation or the relatively inactive ones could be the precursors of the proliferating cells of lentigo maligna.

Enhanced Susceptibility of Melanocytes to Different Immunologic Effector Mechanisms In Vitro: Potential Mechanisms for Postinflammatory Hypopigmentation and Vitiligo

In postinflammatory hypopigmentation and in vitiligo, one observes histologic evidence of melanocyte damage, disappearance of melanocytes, and clinical loss of pigmentation. In the case of vitiligo, this loss of pigment is complete. There is considerable evidence that melanocytes are highly susceptible to autocytotoxic damage and perhaps to specific immunologic damage. We directly compared the susceptibility of cultures of melanocytes (M), keratinocytes (K), endothelial cells (EC), and fibroblasts (F) to hydrogen peroxide damage across a Wide range of concentrations (10‐7‐10‐2 M) and analyzed the differences by computerized Probit analysis. Cytotoxicity was measured by three dye techniques: acridine orange/ethidium bromide (AO/EB), fluorescein diacetate (FD), and nigrosin (N). All three assays produced similar results. The order of susceptibility to H202 was M < EC < K < F. The LD50 of melanocyte targets was two orders of magnitude lower than that of fibroblasts. The AO/EB assay was used to study immunologic cytotoxicity of melanocytes in the presence of sera from vitiligo patients plus either complement or cellular effectors of antibody‐dependent cellular cytotoxicity (ADCC). Eleven vitiligo sera and 11 control sera were contrasted in 4‐ and 16‐hr cytotoxicity assays. Vitiligo patients' sera containing antimelanocyte antibodies induced both complement lysis and ADCC of melanocytes. Thus the melanocyte is highly susceptible to peroxide‐induced damage, complement lysis, and ADCC. In addition, antibodies in vitiligo sera appear to be an important trigger of melanocyte damage by complement and ADCC effectors and are likely to be involved in the melanocyte damage observed in vitiligo.

Detection of DNA-Psoralen Photoadducts in Mammalian Skin

An immunofluorescence (IF) method for the detection of 8-methoxypsoralen (8-MOP) photoadducts to DNA has been developed to assess nuclear damage in keratinocytes and melanocytes after psoralen plus UVA (PUVA) treatment, both under in vitro and in vivo conditions. Cryostat sections of the albino and pigmented guinea pig and human skin were used for in vitro studies to establish minimal and maximal drug concentration and UVA dosimetry for the detection of DNA-8-MOP photoadducts. Limits of detection were as low as 10 ng/cm2 8-MOP and 1 J/cm2 UVA for skin sections and sodium bromide-split epidermal sheets. Guinea pigs treated with topical PUVA revealed positive IF stain in epidermal cell nuclei at a threshold dose of 100 micrograms/cm2 8-MOP and 13 J/cm2 UVA. Pretreatments of cryostat cuts with ethanol and alkali before IF test enhanced the sensitivity of detection in vivo about 10-fold and enabled us to follow the repair of DNA damage after treating normal guinea pig skin with a dose of 50 micrograms/cm2 8-MOP plus 6 J/cm2 UVA. The most interesting findings were as follows: A sensitive method to detect PUVA-induced nuclear damage in epidermal and dermal cells was developed. PUVA treatment induced nuclear DNA damage to melanocytes as well as to adjacent keratinocytes, and melanocytes appeared to be 10 times less vulnerable to photo-damage than keratinocytes. There was a greater propensity for the proliferative cells to be damaged by PUVA. PUVA induced nuclear damage up to 700 micron depth in the dermis. The usefulness of the IF test in detecting DNA damage in microgram and ng amounts in vivo and in following the repair of damaged DNA induced by PUVA.

The epidermal melanocyte population in the skin of ultraviolet-irradiated crested newt.

The response of the epidermal melanocyte population to repeated ultraviolet (UV) exposure (wavelength spectrum 275-350 nm) has been investigated in the crested newt, Triturus cristatus carnifex. The effects of different doses of UV light were studied. The animals were killed 7 months after the first UV exposure. Only a slight decrease in the number of pigment cells was found after 85 sequential irradiations with a total dose of 1.3 x 10(5) J/m2, whereas striking decreases were observed when the same total dose was fractionated into 14 exposures or when a double dose was given in 57 exposures. The relationship between the square roots of the epidermal melanocyte densities and single doses appeared to be roughly linear, at least over the range of doses administered. The main factor in melanocyte damage seemed to be the single dose of irradiation rather than the cumulative dose administered. Decreased melanin content of the keratinocytes was observed in most irradiated animals.

The Effects of UVB and 7, 12, Dimethylbenz(α)anthracene (DMBA) on Epidermal Melanocytes of the Tail in C57BL Mice

The morphological and numerical changes in the epidermal melanocyte system of the tail of C57BL mice were studied after exposure to 7, 12 dimethylbenz(alpha)anthracene (DMBA) followed by UVB irradiation. The dorsal aspect of the tail was exposed for 5 days per week, for a total duration of 10 weeks, to a daily dosage of 0.1 J/cm2 UVB (peak 310 nm). Once a week, 0.2 ml of 0.15% DMBA in acetone was locally applied to the irradiated areas. Biopsies were studied by the combined skin-splitting DOPA and electron microscopic techniques. After 10 weeks of DMBA treatment the following changes were observed: DMBA treatment the following changes were observed: the original brick-like arrangement of melanocytes became confluent, melanocytes were irregularly shaped, dendrites shortened and clumped together, and the outer root sheaths of the hair follicles became covered with melanocytes. There was a significant increase in the number of DOPA positive melanocytes at the end of the first week in DMBA and DMBA + UVB treated skin. Ultrastructurally, an increase in melanosome formation in melanocytes and transfer into keratinocytes was found, as well as redistribution of melanosomes from singlets and doublets into larger groups. Damage of melanocytes by the DMBA treatment was seen, but no inflammation or tumor formation was observed.