Melanin Synthesis In Melanocytes Research Articles

Primary Cilia Negatively Regulate Melanogenesis in Melanocytes and Pigmentation in a Human Skin Model.

The primary cilium is an organelle protruding from the cell body that senses external stimuli including chemical, mechanical, light, osmotic, fluid flow, and gravitational signals. Skin is always exposed to the external environment and responds to external stimuli. Therefore, it is possible that primary cilia have an important role in skin. Ciliogenesis was reported to be involved in developmental processes in skin, such as keratinocyte differentiation and hair formation. However, the relation between skin pigmentation and primary cilia is largely unknown. Here, we observed that increased melanogenesis in melanocytes treated with a melanogenic inducer was inhibited by a ciliogenesis inducer, cytochalasin D, and serum-free culture. However, these inhibitory effects disappeared in GLI2 knockdown cells. In addition, activation of sonic hedgehog (SHH)-smoothened (Smo) signaling pathway by a Smo agonist, SAG inhibited melanin synthesis in melanocytes and pigmentation in a human skin model. On the contrary, an inhibitor of primary cilium formation, ciliobrevin A1, activated melanogenesis in melanocytes. These results suggest that skin pigmentation may be regulated partly by the induction of ciliogenesis through Smo-GLI2 signaling.

Endothelin-1 increases melanin synthesis in an established sheep skin melanocyte culture.

The aims of the study were to establish a culture system for sheep skin melanocytes and uncover the effects of endothelin-1 on melanin synthesis in cultured melanocytes in order to provide an optimal cell system and a theoretical basis for studying the regulatory mechanism of coat color in sheep. In this study, skin punch biopsies were harvested from the dorsal region of 1-3-yr-old sheep, and skin melanocytes were then obtained by the two-step digestion using dispase II and trypsin/ethylene diamine tetraacetic acid (EDTA). The primary cultures of the melanocytes were established and characterized by dopa-staining, immunocytochemical localization of melanocyte markers, and RT-polymerase chain reaction (PCR) analysis of coat color genes. To determine the effect of endothelin-1 on proliferation and melanin synthesis of melanocytes, the cultured cells were treated with different doses of endothelin-1 (10(-7), 10(-8), 10(-9), 10(-10), and 0mol/L), and the growth rate of melanocytes, production of melanin, expression of related genes, and location of related protein in cultured cells were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), ultraviolet spectrophotometry, qRT-PCR, and immunocytochemical localization, respectively. The results showed that the established melanocyte culture functions properly. Endothelin-1 treatment increased markedly the number of melanocytes and melanin content. In responding to this treatment, expressions of microphthalmia-associated transcription factor (MITF), melanocortin 1 receptor (MC1R), tyrosinase (TYR), and endothelin receptor B (EDNRB) in the melanocytes were significantly up regulated (P < 0.05). Immunocytochemical localization revealed that TYR was mainly localized in the cytoplasm. Positive staining of TYR in the melanocytes was significant. The findings demonstrated that the culture system of sheep skin melanocytes was established successfully in vitro, and endothelin-1 promotes the melanogenesis in sheep skin melanocytes.

Singlet oxygen generation during the oxidation of L-tyrosine and L-dopa with mushroom tyrosinase

The generation of singlet oxygen during the oxidation of tyrosine and L-dopa using mushroom tyrosinase in a phosphate buffer (pH 7.4), the model of melanin synthesis in melanocytes, was examined. The reaction was performed in the presence of 2,2,6,6-tetramethyl-4-piperidone (4-oxo-TEMP), an acceptor of singlet oxygen and the electron spin resonance (ESR) of the spin adduct, 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (4-oxo-TEMPO), was measured. An increase in the ESR signal attributable to 4-oxo-TEMPO was observed during the oxidation of tyrosine and L-dopa with tyrosinase, indicating the generation of singlet oxygen. The results suggest that 1O2 generation via tyrosinase-catalyzed melanin synthesis occurs in melanocyte.

Benzo(a)pyrene represses melanogenesis in B16F10 mouse melanoma cells

Benzo(a)pyrene (BaP) is a chemically based polycyclic aromatic hydrocarbon (PAH) that is readily absorbed by the skin. BaP is metabolized to BaP-diol-epoxide by cytochromes P-450 1A1/2 (CYP1A1/2) and cytochromes P-450 1B1 (CYP1B1) in the cytosol. BaP and its metabolites induce genotoxicity and cancer. Although BaP easily accumulates in melanin-containing tissues as well as other tissue types, the effects of BaP on melanocytes are not fully understood. Here, we show that 40-100 µM BaP represses melanin synthesis in B16F10 cells. The decrease of melanin contents is induced by tyrosinase activity in BaP-exposed B16F10. However, this repression of melanin synthesis is not induced by direct inhibition of tyrosinase in in vitro assay. Therefore, we show whether BaP regulated melanin synthesis-related enzyme. BaP regulates melanin synthesis by Tyr and Tyrpl expression. In addition, these genes expression is down-regulated by Mitf repressed by BaP. Importantly, the repression was provoked in the absence and presence of α-melanocyte stimulating hormone (α-MSH). Therefore, we hypothesize BaP interrupts the UV protection mechanism by repressing melanin synthesis in the skin. Taken together our results have revealed new side effects that exposure of BaP abolished melanin synthesis in melanocytes.

Unrevealing the role of P-protein on melanosome biology and structure, using siRNA-mediated down regulation of OCA2.

The pink-eyed dilution protein (P-protein) plays a critical role in melanin synthesis in melanocytes and retinal pigment epithelium cells. Mutation in this protein may cause complete or partial albinism. Role of the P-protein ranges in melanin synthesis to maturation and trafficking of the melanosomes. The aim of the present study was to evaluate the effect of P-protein inhibition on melanosome biology by comparing the shape, size, count, and types of melanosomes in melan-a melanocytes. The cells were extensively examined by the transmission electron microscopy. The P-protein inhibition was carried by P-protein-siRNA transfection to melan-a melanocytes, B16F10 mouse melanoma, and melan-p1 cells. Measurement of melanin contents, cellular tyrosinase, and different tyrosinase related proteins were also determined to investigate the effect of P-protein siRNA transfection on melanocytes. Results suggested that the inhibition of P-protein can significantly change the melanosomal morphology, types and their respective numbers, and provided a novel strategy for the control of melanin synthesis.

Ardisia crenata extract stimulates melanogenesis in B16F10 melanoma cells through inhibiting ERK1/2 and Akt activation.

Melanin protects the skin against ultraviolet radiation by scattering incoming light and absorbing diverse free radicals. Agents that increase melanin synthesis in melanocytes may reduce the risk of photodamage and skin cancer. The present study investigated the effect of a methanol extract of Ardisia crenata (AC) on melanogenesis in B16F10 cells. Treatment of cultured B16F10 cells with AC extract (10, 20 and 40 µg/ml) stimulated an increase in melanin levels in a concentration-dependent manner, without cytotoxicity. Tyrosinase is key in the regulation of melanin production, thus the effect of AC extract on tyrosinase activity and protein expression was analyzed. AC extract was observed to significantly increase tyrosinase activity and protein expression in B16F10 cells. Furthermore, AC extract was found to markedly increase the protein expression of microphthalmia-associated transcription factor, which is an important transcription factor involved in tyrosinase gene expression. In addition, AC extract (40 µg/ml) was observed to suppress the activation of extracellular signal-regulated kinase (ERK) and Akt, which negatively regulate melanin synthesis in B16F10 cells. In conclusion, to the best of our knowledge, the present study is the first to show that a methanol extract of AC stimulates melanogenesis by increasing tyrosinase expression via the inhibition of ERK and Akt. Thus, methanol extract of AC may be a potential treatment for hypopigmentation diseases and may be a candidate for skin-tanning cosmetic products.

Inulavosin and its benzo‐derivatives, melanogenesis inhibitors, target the copper loading mechanism to the active site of tyrosinase

Tyrosinase, a melanosomal membrane protein containing copper, is a key enzyme for melanin synthesis in melanocytes. Inulavosin inhibits melanogenesis by enhancing a degradation of tyrosinase in lysosomes. However, the mechanism by which inulavosin redirects tyrosinase to lysosomes is yet unknown. The analyses of structure-activity relationship of inulavosin and its benzo-derivatives reveal that the hydroxyl and the methyl groups play a critical role in their inhibitory activity. Intriguingly, the docking studies to tyrosinase suggest that the compounds showing inhibitory activity bind through hydrophobic interactions to the cavity of tyrosinase below which the copper-binding sites are located. This cavity is proposed to be required for the association with a chaperon that assists in copper loading to tyrosinase in Streptomyces antibioticus. Inulavosin and its benzo-derivatives may compete with the copper chaperon and result in a lysosomal mistargeting of apo-tyrosinase that has a conformational defect.

Geranylgeranylacetone inhibits melanin synthesis via ERK activation in Mel-Ab cells

AimsGeranylgeranylacetone (GGA) has shown cytoprotective activity through induction of a 70-kDa heat shock protein (HSP70). Although HSP70 is reported to regulate melanogenesis, the effects of GGA on melanin synthesis in melanocytes have not been previously studied. Therefore, this study investigated the effects of GGA on melanogenesis and the related signaling pathways. Main methodsMelanin content and tyrosinase activities were measured in Mel-Ab cells. GGA-induced signal transduction pathways were investigated by western blot analysis. Key findingsOur results showed that GGA significantly decreased melanin content in a concentration-dependent manner. Similarly, GGA reduced tyrosinase activity dose-dependently, but it did not directly inhibit tyrosinase. Western blot analysis indicated that GGA downregulated microphthalmia-associated transcription factor (MITF) and tyrosinase protein expression, whereas it increased the phosphorylation of extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin (mTOR). Furthermore, a specific ERK pathway inhibitor, PD98059, blocked GGA-induced melanin reduction and then prevented downregulation of MITF and tyrosinase by GGA. However, a specific mTOR inhibitor, rapamycin, only slightly restored inhibition of melanin production by GGA, indicating that mTOR signaling is not a key mechanism regulating the inhibition of melanin production. SignificanceThese findings suggest that activation of ERK by GGA reduces melanin synthesis in Mel-Ab cells through downregulation of MITF and tyrosinase expression.

Essential Role of RAB27A in Determining Constitutive Human Skin Color

Human skin color is predominantly determined by melanin produced in melanosomes within melanocytes and subsequently distributed to keratinocytes. There are many studies that have proposed mechanisms underlying ethnic skin color variations, whereas the processes involved from melanin synthesis in melanocytes to the transfer of melanosomes to keratinocytes are common among humans. Apart from the activities in the melanogenic rate-limiting enzyme, tyrosinase, in melanocytes and the amounts and distribution patterns of melanosomes in keratinocytes, the abilities of the actin-associated factors in charge of melanosome transport within melanocytes also regulate pigmentation. Mutations in genes encoding melanosome transport-related molecules, such as MYO5A, RAB27A and SLAC-2A, have been reported to cause a human pigmentary disease known as Griscelli syndrome, which is associated with diluted skin and hair color. Thus we hypothesized that process might play a role in modulating skin color variations. To address that hypothesis, the correlations of expression of RAB27A and its specific effector, SLAC2-A, to melanogenic ability were evaluated in comparison with tyrosinase, using human melanocytes derived from 19 individuals of varying skin types. Following the finding of the highest correlation in RAB27A expression to the melanogenic ability, darkly-pigmented melanocytes with significantly higher RAB27A expression were found to transfer significantly more melanosomes to keratinocytes than lightly-pigmented melanocytes in co-culture and in human skin substitutes (HSSs) in vivo, resulting in darker skin color in concert with the difference observed in African-descent and Caucasian skins. Additionally, RAB27A knockdown by a lentivirus-derived shRNA in melanocytes concomitantly demonstrated a significantly reduced number of transferred melanosomes to keratinocytes in co-culture and a significantly diminished epidermal melanin content skin color intensity (ΔL* = 4.4) in the HSSs. These data reveal the intrinsically essential role of RAB27A in human ethnic skin color determination and provide new insights for the fundamental understanding of regulatory mechanisms underlying skin pigmentation.

Evaluation of the inhibition of mushroom tyrosinase and cellular tyrosinase activities of oxyresveratrol: comparison with mulberroside A

The inhibitory effects of oxyresveratrol, the aglycone of mulberroside A, on mushroom and cellular tyrosinase activities and melanin synthesis were evaluated. Mulberroside A and oxyresveratrol showed inhibitory activity against mushroom tyrosinase, with oxyresveratrol demonstrating a greater inhibitory effect than that of mulberroside A. Oxyresveratrol and mulberroside A strongly inhibited melanin production in Streptomyces bikiniensis and exhibited dose-dependent inhibition of tyrosinase activity and inhibition of melanin synthesis in B16F10 melanoma cells. However, the compounds exhibited nearly similar inhibitory effects on the activity of cellular tyrosinase and melanin synthesis in murine melanocytes. The inhibition of melanin synthesis by mulberroside A and oxyresveratrol was involved in suppressing the expression level of melanogenic enzymes, tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2). These results indicate that the inhibition rate of mushroom tyrosinase might not provide an accurate estimate of the inhibition rate of melanin synthesis in melanocytes.

5,7-Dimethoxyflavone induces melanogenesis in B16F10 melanoma cells through cAMP-dependent signalling

Melanin protects the skin against ultraviolet radiation (UVR) and diverse free radicals. Agents that increase melanin synthesis in melanocytes may reduce UVR-induced skin damage and skin cancer. In the present study, we evaluated the effects of 5,7-dimethoxyflavone (5,7-DMF) on melanogenic protein expression and signalling pathways. We found that 5,7-DMF significantly increased melanin content by upregulating microphthalmia-associated transcription factor and related melanogenic proteins. Additionally, 5,7-DMF increased cAMP levels, which activates a cascade of reactions, such as cAMP responsive element-binding protein and Akt/glycogen synthase kinase-3β (GSK-3β) signalling. Thus, 5,7-DMF may be an effective pigmentation stimulator for photoprotection and hypopigmentation disorders.

Inhibition of basal and ultraviolet B-induced melanogenesis by cannabinoid CB1 receptors: a keratinocyte-dependent effect

Ultraviolet radiation is the major environmental insult to the skin and stimulates the synthesis of melanin in melanocytes, which then distribute it to the neighboring keratinocytes where it confers photo-protection. Skin color results from the paracrine interaction between these two cell types. Recent studies suggest that endocannabinoids are potential mediators in the skin. Here, we investigated whether cannabinoid drugs play a role in melanogenesis and if ultraviolet radiation modifies the cutaneous endocannabinoid system. We used human melanotic melanoma cell line (SK-mel-1) in monoculture or co-culture with human keratinocytes (HaCat). Endocannabinoid levels, cannabinoid receptors expression, and melanin content were evaluated under basal conditions and after ultraviolet-B irradiation (311 nm). We provide evidence that human melanoma cells (SK-mel-1) express CB(1) receptors, and when in co-culture with keratinocytes (HaCat), the selective CB(1) receptor agonist arachidonyl-2-chloroethylamide (ACEA 1 and 10 μM) inhibited (by 33.4 and 37.3%, respectively) basal melanogenesis. In addition, ultraviolet-B-induced melanogenesis in co-cultures was abolished by ACEA 10 μM. Both ACEA inhibitory effects were reversed by AM251 (1 μM), a selective CB(1) antagonist. Furthermore, ultraviolet-B radiation increased endocannabinoids levels only in keratinocytes, whereas CB(1) cannabinoid receptor expression was up-regulated only in melanoma cells. Our results collectively suggest that ultraviolet radiation activates paracrine CB(1)-mediated endocannabinoid signaling to negatively regulate melanin synthesis. The endocannabinoid system in the skin may be a possible target for future therapies in pigmentary disorders.

Effect of Natural Plant Water Extracts Suppressed Melanin Production In Human skin cell(CCD‐986sk)

Melanin is the pigment responsible for the color of human skin. Tyrosinase is known to be a key enzyme that catalyzes the synthesis of melanin in melanocytes. Therefore, we intend to verify the effect of whitening using these natural plant water extract (MH: Ephedrae, Santalum album, BDH: Agrimonia pilosa ledeb,SHC: Houttuyniae herbal, ESC: Houttuynia cordata Thunberg, JGP: Sorbus amurensis Koehne, TSJ: Coscuta japonica chois ). The effect of natural water extracts on antioxidant activities, tyrosinase activity, melanin biosynthesis have been studied in a human skin cells or in a cell‐free system. The tyrosinase activity and melanin content was assessed using the RT‐RCR, western blotting and spectrophotometric methods. The results show that the SHC has a greater ability to inhibit tyrosinase activity than tested five extracts. MH, SHC, and JGP significantly suppressed melanin production in a human skin cells.(Following are results of a study on the “Human Resource Development Center for Economic Region Leading Industry” Project, supported by the Ministry of Education, Science &amp; Technology(MEST) and the National Research Foundation of Korea(NRF).)

Effect of calcipotriol on melanin synthesis by human melanocytes.

Objective To investigate the effect of calcipotriol on melanin synthesis by human melanocytes and its possible action mechanism.Methods Primary melanocytes were cultured with various concentrations(10-5,10-6,10-7,10-8,10-9,10-10 mol/L)of calcipotriol for 24 or 48 hours.Subsequently,MTT assay,NaoH assay.Dopa-oxidase assay,Western blot and semiquantitative RT-PCR were used to measure the cell proliferation of,melanin synthesis by.tyrosinase activity,protein and mRNA expression levels in the melanocytes.respectively.Those untreated melanocytes served as the control.Results The calcipotriol between 10-9 and 10-5 mol/L had no significant effect on the proliferation of cultured melanocytes(P＞0.05).while that of 10-9 and 10-8 mol/L increased tyrosinase activity by 137%and 123%,and enhanced melanin synthesis by 40.63%and 18.75%,respectively,compamd with untreated melanocytes(both P＜0.05).Moreover,the tyrosinase protein level increased by 270.4%(P＜0.05)in melanocytes treated with calcipotriol at 10-9 mol/L for 24 hours.The strongest tyrosinase activity and melanin synthesis was observed in melanocytes treated with calcipotriol of 10-9 moI/L.Conclusions The proliferation of melanocytes is unaffected by calcipotriol at 10-9 to 10-5 mol/L,but it can elevate the expression of tyrosinase protein,enhance tyrosinase activity,and promote melanin synthesis in melanocytes. Key words: Melanocytes; Melanins; Calcitriol; Monophenol monooxygenase

Inulavosin, a Melanogenesis Inhibitor, Leads to Mistargeting of Tyrosinase to Lysosomes and Accelerates its Degradation

The melanosome is a highly specialized organelle where melanin is synthesized. Tyrosinase and tyrosinase-related protein-1 (Tyrp1) are major melanosomal membrane proteins and key enzymes for melanin synthesis in melanocytes. Inulavosin, a melanogenesis inhibitor isolated from Inula nervosa (Compositae), reduced the melanin content without affecting either the enzymatic activities or the transcription of tyrosinase or Tyrp1 in B16 melanoma cells. To our knowledge, this inhibitor is previously unreported. Electron-microscopic analyses revealed that inulavosin impaired late-stage development of melanosomes (stages III and IV), in which melanin is heavily deposited. However, it did not alter the early stages of melanosomes (stages I and II), when filamentous structure is observed. Immunofluorescence analyses showed that tyrosinase, but not Tyrp1, was specifically eliminated from melanosomes in cells treated with inulavosin. Unexpectedly, inulavosin specifically accelerated the degradation of tyrosinase but not other melanosomal/lysosomal membrane proteins (Tyrp1, Pmel17, and LGP85). The degradation of tyrosinase induced by inulavosin associated with lysosomes but not the proteasome. Interestingly, lysosomal protease inhibitors restored the melanogenesis but not the targeting of tyrosinase to melanosomes in the cells treated with inulavosin. Instead, colocalization of tyrosinase with lysosome-associated membrane protein-1 at late endosomes/multivesicular bodies and lysosomes was accentuated. Taken together, inulavosin inhibits melanogenesis as a result of mistargeting of tyrosinase to lysosomes.

The natural plant water extracts suppressed melanin production in murine

Melanin is the pigment responsible for the color of human skin. Tyrosinase is known to be a key enzyme that catalyzes the synthesis of melanin in melanocytes. The effects of natural plant water extracts [(Camellia sinensis (BIC), Black,allium sativum. L (HMN), Aloe (ALE), Crataegus pinnatifida (SS), Paeonia suffruticosa (MDP), Trichosanthes kirilowii Maxim (CHB), Cornus officinalis (SSY), Discorea japonica Thunb (SY)) on antioxidant, the activity of mushroom tyrosinase and melanin synthesis have been studied in a cell‐free system and mouse melanoma B16 cell. Radical‐scavenging activity was tested as reactivity towards 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH). The results showed that all the extracts exhibited antioxidant activity and the MDP, BIC and SSY extracts possessed high antioxidant among eight water extracts. The cellular tyrosinase activity and melanin content measured in mouse melanoma B16 cell by RT‐PCR and Western blot analysis. Treatment with HMN and ALE extracts significantly inhibited melanogenesis by tyrosinase. The melanin content of melanoma B16 cell was significantly decreased by treatment with MDP, BIC, HMN and ALE water extracts. Our results indicate that MDP, BIC, HMN and ALE water extracts exhibited antioxidant activity, tyrosinase and melanin reducing activities. The natural extracts may be a candidate for cosmetic use.

Inhibitory effects of arbutin on melanin biosynthesis of α-melanocyte stimulating hormone-induced hyperpigmentation in cultured brownish guinea pig skin tissues

Arbutin has been used as a whitening agent in cosmetic products. Melanin, the major pigment that gives color to skin, may be over-produced with sun exposure or in conditions such as melasma or hyperpigmentary diseases. Tyrosinase is a key enzyme that catalyzes melanin synthesis in melanocytes; therefore, inhibitors of the tyrosinase enzyme could be used for cosmetic skin whitening. A recent study has reported that arbutin decreases melanin biosynthesis through the inhibition of tyrosinase activity. However, this inhibitory mechanism of arbutin was not sufficiently demonstrated in skin tissue models. We found that arbutin both inhibits melanin production in B16 cells induced with alpha-MSH and decreases tyrosinase activity in a cell-free system. Furthermore, the hyperpigmentation effects of alpha-MSH were abrogated by the addition of arbutin to brownish guinea pig and human skin tissues. These results suggest that arbutin may be a useful agent for skin whitening.

The expanding role and presence of neuromelanins in the human brain – why gray matter is gray

The expanding role and presence of neuromelanins in the human brain – why gray matter is gray

SOX9 and the tanning response: something new under the sun

SOX9 and the tanning response: something new under the sun

Commentary 5

Studies on the pathogenesis focussing on melanocyte biology, melanin biochemistry or skin immunity have yielded a variety of hypotheses to explain the disappearance of melanocytes in vitiligo, including a genetic predisposition, increased oxidative stress and toxic metabolites, neurochemical factors and autoimmunity (1). Whereas these factors probably all contribute to the development of depigmentation (convergence theory) (2), their interaction is not clearly defined. This commentary presents a multidisciplinary view of the pathogenesis of vitiligo (Fig. 1). Schematic overview of the pathogenesis of vitiligo. Melanin synthesis in melanocytes is a tightly regulated process. Defects in the protective mechanism in the skin that scavenge radicals and toxic intermediates of melanin production may lead to vitiligo. The increased levels of oxidative stress in the vitiligo skin leads to the deactivation of catalase (3). H202 can also oxidize (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin to 6-biopterin, which is cytotoxic to melanocytes. Although this melanocyte death may account for part of the depigmentation, dying melanocyte fragments will be taken up by epidermal Langerhans cells. The uptake of self-antigens from dying melanocytes in the absence of activation signals does not activate the Langerhans cells and will therefore not lead to immunity against melanocytes. Concurrent external stress factors, such as wounding, high dose of UV radiation or hormonal changes, however, activate Langerhans cells and dendritic cells in the skin, leading to breakage of tolerance (4). Clinically, these stress factors induce progression of depigmentation. The Koebner phenomenon results from stress factors at remote skin sites that induce local activation of Langerhans cells and reactivation of anti-melanocyte immunity, leading to the formation of new lesions. The lower threshold for breakage of tolerance in patients with vitiligo is illustrated by the increased incidence of autoimmune diseases in patients with vitiligo (5-12). Normally, regulatory T cells maintain peripheral tolerance by suppressing autoreactive T-cell activity. In patients with vitiligo, however, decreased levels of regulatory T cells in the skin lower the threshold for autoimmunity. Activated Langerhans cells containing melanocyte antigens migrate to the lymph nodes and present melanocyte antigenic peptides bound to HLA molecules to T cells. The association of vitiligo vulgaris with certain HLA class II alleles (13-15) indicates the dominance of peptides binding to these HLA types in inducing immunity. In the lymph nodes, melanocyte-reactive CD8+ and CD4+ T cells are activated to proliferate and migrate to the skin, resulting in increased levels of T cells reactive with tyrosinase, gp100 or MART-1 in peripheral blood (16-19), as well as autoantibodies against these antigens (20-22). Moreover, the number of circulating MART-1-reactive T cells expressing CLA in patients with vitiligo correlated with the extent of depigmentation (18). In perilesional skin, CD8+ T cells, macrophages and to a lesser extent CD4+ T cells were found (23, 24). Infiltrating CD8+ T cells expressed skin homing, cutaneous leukocyte-associated antigen (CLA) and T-cell activation markers CD25, perforin and granzyme B (25), and colocalized with disappearing melanocytes. Finally, perilesional melanocyte-reactive CD8+ cytotoxic T cells were shown to be capable of actively killing melanocytes in autologous skin tissue (J.G. van den Boorn, D. Konijnenberg, T.A.M. Dellemijn, J.P.W. van der Veen, J.D. Bos, C.J.M. Melief, F.A. Vyth-Dreese and R.M. Luiten, unpublished data). Taken together, the pathogenesis of vitiligo results from changes in biochemical processes in the skin that trigger autoimmunity, which is enhanced by genetic predisposition to autoimmunity.