Study of CCN3 (NOV) and DDR1 in normal melanocytes and vitiligo skin

We have hypothesised that melanocytes disappear in vitiligo because they are weakly attached to the epidermal basal membrane (melanocytorrhagy). In the epidermis, attachment of melanocytes to collagen IV is mediated through DDR1, which is under the control of CCN3. DDR1 genetic variants have been associated with vitiligo in patients of different ethnic origin. In vitro studies have shown that inhibition of CCN3 induces the detachment of melanocytes. We have studied in parallel the expression of CCN3 and DDR1 in lesional and perilesional skin of patients with vitiligo and the impact of the silencing of CCN3 and DDR1 in normal human melanocytes on their behaviour in epidermal reconstructs. Our in vivo study provides evidence of a dysregulation of the DDR1-CCN3 interaction in vitiligo skin as melanocytes remaining in perilesional skin did not express CCN3. Expression of DDR1 was decreased in lesional versus perilesional vitiligo skin in the majority of patients, and the expression of collagen IV was found decreased in all patients. Silencing of CCN3 in melanocytes induced a significant inhibition of cell adhesion to collagen IV whereas melanocytes transduced with shDDR1 still adhered well on collagen IV and did not increase melanocyte loss in epidermal reconstructs as compared with normal melanocytes. Melanocyte detachment was observed but not in all reconstructs using CCN3 silenced melanocytes. Overall, our study confirms that a downregulation of CCN3 is implicated in melanocyte adhesion in part through DDR1. In vitiligo skin, the interaction of CCN3 with other molecules, such as TGFβ and CCN2, needs to be addressed.     

Melanocytorrhagy and apoptosis in vitiligo: Connecting jigsaw pieces

Vitiligo is an acquired depigmenting disorder characterized by a chronic and progressive loss of melanocytes from the epidermis and follicular reservoir. The mechanism of melanocyte disappearance has never been clearly understood. This review discussed the data supporting the theory of melanocytorrhagy and apoptosis as one of the primary defects underlying melanocyte loss. Theory of melanocytorrhagy proposes that non-segmental vitiligo is a primary melanocytorrhagic disorder with altered melanocyte responses to friction and possibly other types of stress, inducing their detachment and subsequent transepidermal loss. Melanocytes detachment induces apoptosis whereas adherence to basement membrane suppresses apoptosis. The study of apoptosis, mechanisms of its induction, and the ways to block apoptosis is one possible way to find both the causes of depigmentation and medications to prevent its progression.     

The expression of the c-kit receptor by epidermal melanocytes may be reduced in vitiligo

Summary 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 basai ceils) 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 ceils 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.     

白癜风皮损边缘黑素细胞线粒体超微结构的电镜观察

【摘要】 目的 探讨白癜风患者皮损边缘黑素细胞线粒体结构的变化。方法 在透射电镜下观察健康对照、进展期白癜风及稳定期白癜风患者皮损边缘黑素细胞形态,体视学方法测量线粒体体密度（Vv）、表面积密度（Sv）、数密度（Nv）等参数。结果 健康对照组黑素细胞可见大量黑素小体（28.57 ± 3.21）,以Ⅲ、Ⅳ期为主,线粒体规则分布在细胞内,结构正常、嵴密集,部分细胞胞质内可见自噬小体。进展期和稳定期白癜风黑素细胞内黑素小体数量减少,单位细胞内黑素小体数量分别为22 ± 6.16和17.43 ± 6.24,其中,Ⅲ期黑素小体显著减少,线粒体大小不一、形态多样,大部分线粒体明显肿胀,嵴模糊、排列紊乱甚至断裂,呈空泡状改变,未见线粒体自噬现象。线粒体形态结构定量研究显示,健康对照组Nv、Vv、Sv分别为（7.194 ± 1.434） μm－3、（4.8 ± 1.2）%、（2.42 ± 0.86） μm－1;进展期白癜风组Nv、Vv、Sv分别为（4.055 ± 0.906） μm－3、（7.4 ± 2.1）%、（3.58 ± 1.15） μm－1;稳定期白癜风组Nv、Vv、Sv分别为（5.311 ± 0.873） μm－3、（6.5 ± 1.4）%和（2.82 ± 0.94） μm－1,组间差异有统计学意义（P ＜ 0.05）。结论 白癜风皮损边缘黑素细胞线粒体受损,且进展期损伤程度大于稳定期。 【关键词】 白癜风; 黑素细胞; 线粒体; 显微镜检查,电子,透射     

Defective calcium transport in vitiliginous melanocytes

Melanocytes were successfully established from involved and uninvolved skin of a patient with acute acrofacial vitiligo. Cells from involved epidermis showed a fivefold decrease in the rate of radiolabelled⁴⁵Ca uptake compared with uninvolved and control cells. These results are similar to previous findings in keratinocytes from involved skin in patients with vitiligo. Since 6-biopterin is cytotoxic to melanocytes and calcium controls the redox status of the 6-biopterin/(6R)5, 6, 7, 8-tetrahydrobiopterin equilibrium via the thioredoxin reductase/thioredoxin system, these results underline the importance of this electron transfer system for both melanocyte function and survival.     

同种异体淋巴细胞与黑素细胞混合培养的实验研究

目的：黑素细胞与同种异体淋巴细胞混合培养,从体外实验来研究排斥反应的可能性。方法：采用^3H－dTR掺入同位素液态闪烁计数法测定淋巴细胞的转化增殖率,并用透射电镜观察混合培养后黑素细胞的超微结构。结果：黑素细胞对同种异体淋巴细胞的转化增殖率与ConA刺激淋巴细胞转化增殖的阳性对照比较,结果显示黑素细胞的促淋巴细胞转化增殖的特异性抗原作用较弱。进一步比较黑素细胞对不同病期白癜风患者淋巴细胞的影响,发现黑素细胞对活动期白癜风患者淋巴细胞的刺激作用相对较强,而稳定期患者和正常人对照组结果差异无显著性。同时电镜观察结果显示同种异体的淋巴细胞对黑素细胞无明显损伤,黑素细胞经混合培养后,细胞的形态完整,黑素合成功能正常,胞体和树突正损伤。细胞内细胞器丰富,胞浆内可见大量的不同阶段黑素小体。结论：正常人黑素细胞的促淋巴细胞转化增殖的异性抗原作用较弱。同种异体淋巴细胞对黑素细胞超微结构无破坏作用。在进行同种异体移植时最好选择稳定期患者。     

BIOLOGIC CHARACTERISTICS OF CULTURED HUMAN VITILIGO MELANOCYTES

Background. Vitiligo is a pigmentary disorder of unknown cause characterized by depigmented patches due to destruction of melanocytes. Recently, the inherent cellular defect theory has been discussed. To investigate the biologic characteristics of cultured melanocytes from normal and vitiligo subjects, this study had the purpose to examine the functional and ultrastructural characteristics of these melanocytes and to observe the morphologic and functional changes of melanocytes in response to ultraviolet B irradiation. Methods. Melanocytes were isolated and cultured from foreskin and arm skin of normal and vitiligo subjects. The DNA synthesis, tyrosinase activity assay, transmission and scanning electron microscopic examination, and the effects of ultraviolet B(uvB)-irradiation on cultured melanocytes were studied. Results. Vitiligo melanocytes showed no significant differences in DNA synthesis and tyrosinase activity compared with normal melanocytes, but the vitiligo melanocytes contained dilated and/or circular rough endoplasmic reticulum (RER) on transmission electron microscopic examination. Exposure of the cultured melanocytes to UVB resulted in increased protein synthesis and tyrosinase activity. Morphologic alterations and changes in DNA synthesis were also noted. Compared with normal melanocytes, the responses of vitiligo melanocyte to UVB showed no significant difference. Conclusions. Normal and vitiligo melanocytes showed similar biologic characteristics except in the changes of RERS in the vitiligo melanocytes. The ultrastructural aberrations in vitiligo subjects do not seem to be directly related to the biologic characteristics and the responses to UVB irradiation in vitiligo melanocytes.     

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.     

Growth defects of melanocytes in culture from vitiligo subjects are spontaneously corrected in vivo in repigmenting subjects and can be partially corrected by the addition of fibroblast-derived growth factors in vitro

Summary Melanocytes cultured from uninvolved skin of untreated vitiligo subjects have decreased initial seeding capacities, manifest a lag period for the onset of the growth phase, and cannot be passaged. In contrast, melanocytes obtained from uninvolved and perilesional skin of vitiligo subjects actively repigmenting under 8-methoxy psoralen plus sunlight (PUVA) therapy have higher initial seeding capacities, grow faster without a lag period, and can be passaged to more than 12 passages. Extracts of a fetal lung fibroblast cell line (PMR-GF) that promote the growth rates and passage capacities of melanocytes from normal adult donors have been found also to promote the growth rates and passage capacities of melanocytes from the uninvolved skin of vitiligo subjects. Extracts of a fetal lung fibroblast cell line (PMR-GF), however, did not have any further stimulatory effect on the growth of melanocytes obtained from repigmenting vitiligo subjects. Melanocytes cultured from normal and untreated vitiligo subjects grew individually dispersed in the absence of PMR-GF, but tended to grow in clusters in its presence. Melanocytes from the repigmenting vitiligo subjects, however, tended to grow in clusters even in the absence of PMR-GF. These results indicate that the defective in vitro growth characteristics of melanocytes from vitiligo subjects may be related to the pathogenesis of this disease. It is possible that growth factors may be involved in the process of repigmentation in vitiligo subjects.     

Aberrant expression of complement regulatory proteins, membrane cofactor protein and decay accelerating factor, in the involved epidermis of patients with vitiligo

BACKGROUND: Vitiligo is a pigmentary disorder of the skin characterized by the complete absence of melanocytes from the lesion. Complement-activating antimelanocyte antibodies have been implicated in vitiligo pathogenesis. As membrane regulators of complement activation, membrane cofactor protein, decay accelerating factor and CD59 protect cells from elimination by autologous complement, their absence or downregulation on melanocytes may be associated with autoantibody and complement-mediated melanocyte destruction in vitiligo. OBJECTIVES: We studied the expression of these regulatory proteins in non-lesional, perilesional and lesional vitiligo skin compared with those of control specimens. METHODS: We used immunohistochemistry to study the expression of the regulatory proteins, and flow cytometric analysis of cultured melanocytes to investigate possible constitutive changes in the expression levels of these molecules. We also investigated whether melanocytes can influence keratinocyte susceptibility to autologous complement by regulating keratinocytic decay accelerating factor and membrane cofactor protein expression levels. RESULTS: Immunohistochemical data showed that expression of membrane cofactor protein and decay accelerating factor in whole epidermis was lower in lesional and perilesional skin in comparison with non-lesional skin. The reduced in situ expression appeared to be specific to vitiligo. However, coculture experiments indicated that melanocytes do not influence keratinocyte susceptibility to autologous complement. Further, flow cytometric analysis of cultured melanocytes convincingly demonstrated that non-lesional vitiligo and control melanocytes have comparable decay accelerating factor, membrane cofactor protein and CD59 expression levels. CONCLUSIONS: It is therefore concluded that there is no constitutive melanocyte defect per se that could be related to the in vivo expression of these molecules in vitiligo. Nevertheless, the present data suggest that both keratinocytes and melanocytes in the involved vitiliginous whole epidermis express lower levels of decay accelerating factor and membrane cofactor protein compared with controls that could render them more vulnerable to autologous complement attack.     

Defective calcium transport in vitiliginous melanocytes

Melanocytes were successfully established from involved and uninvolved skin of a patient with acute acrofacial vitiligo. Cells from involved epidermis showed a fivefold decrease in the rate of radiolabelled ⁴⁵ Ca uptake compared with uninvolved and control cells. These results are similar to previous findings in keratinocytes from involved skin in patients with vitiligo. Since 6-biopterin is cytotoxic to melanocytes and calcium controls the redox status of the 6-biopterin /(6 R )5, 6, 7, 8-tetrahydrobiopterin equilibrium via the thioredoxin reductase/thioredoxin system, these results underline the importance of this electron transfer system for both melanocyte function and survival. Received: 21 November 1994     

白癜风患者皮损中CD4~+,CD8~+T淋巴细胞及朗格汉斯细胞的检测

目的探讨白癜风患者皮损中CD4+,CD8+T淋巴细胞、朗格汉斯细胞(LC)及黑素细胞(MC)在白癜风发病中的作用。方法采用Envision免疫组化染色法,对12例白癜风进展期患者和9例稳定期患者皮损处CD4+,CD8+T淋巴细胞、LC及MC进行检测,并与10例正常人皮肤进行对照。结果白癜风患者皮损中CD4+,CD8+T淋巴细胞、LC表达较对照组显著增多(P<0.01),而MC表达较对照组显著减少(P<0.01)。结论白癜风患者皮损中LC,CD4+,CD8+T淋巴细胞异常表达可能参与白癜风的发病,其作用模式可能是LC抗原递呈,CD4+,CD8+T淋巴细胞浸润破坏或攻击MC,从而引起白癜风患者表皮基底层的MC减少或消失,导致白癜风的发生。     

Structural aberration of the rough endoplasmic reticulum and melanosome compartmentalization in long-term cultures of melanocytes from vitiligo patients

Long-term cultures of melanocytes were established from 14 subjects with vitiligo and from five normal controls and analyzed ultrastructurally. Cultured melanocytes from 78.6% of the vitiligo patients demonstrated abnormalities that consisted of 1) dilation of the rough endoplasmic reticulum (RER), 2) circular RER profiles, and/or 3) membrane bound compartments of melanosomes. Cultured melanocytes from control subjects were predominantly normal with only one of the normal cultures demonstrating minimal circular RER profiles. The three unique abnormal structures in cultured vitiligo melanocytes were not always concomitantly expressed and could not be associated with any specific clinical feature of vitiligo. Quantitative analysis of the RER demonstrated that the profiles of dilated RER in cultured vitiligo melanocytes expressed a significant 1.5-2.8-times increase in mean cisternal area over cultured control melanocytes (i.e., 5.41-9.92 microns 2 versus 3.53 microns 2, respectively). The cisterna of the dilated RER profiles frequently contained floccular material that appeared to originate from the ribosomes, an indication that the floccular material may be translation products. The dilation of RER in melanocytes from the same patient persisted through repeated subculturing for up to 14.75 months. Epidermal melanocytes in biopsied skin from a patient whose cultured melanocytes were aberrant also demonstrated dilated and circular RER profiles. These results demonstrate that melanocytes from most vitiligo patients express an innate defect when cultured. Although this defect does not appear to be cytotoxic in vitro, this abnormality may be the primary defect that elicits melanocyte destruction in vivo.     

Development of melanocye-keratinocyte co-culture model for controls and vitiligo to assess regulators of pigmentation and melanocytes

Background: There is a need to develop an in vitro skin models which can be used as alternative system for research and testing pharmacological products in place of laboratory animals. Therefore to study the biology and pathophysiology of pigmentation and vitiligo, reliable in vitro skin pigmentation models are required. Aim: In this study, we used primary cultured melanocytes and keratinocytes to prepare the skin co-culture model in control and vitiligo patients. Methods: The skin grafts were taken from control and patients of vitiligo. In vitro co-culture was prepared after culturing primary melanocytes and keratinocytes. Co- cultures were treated with melanogenic stimulators and inhibitors and after that tyrosinase assay, MTT assay and melanin content assay were performed. Results: Melanocytes and keratinocytes were successfully cultured from control and vitiligo patients and after that co-culture models were prepared. After treatment of co-culture model with melanogenic stimulator we found that tyrosinase activity, cell proliferation and melanin content increased whereas after treatment with melanogenic inhibitor, tyrosinase activity, cell proliferation and melanin content decreased. We also found some differences in the control co-culture model and vitiligo co-culture model. Conclusion: We successfully constructed in vitro co-culture pigmentation model for control and vitiligo patients using primary cultured melanocytes and keratinocytes. The use of primary melanocytes and keratinocytes is more appropriate over the use of transformed cells. The only limitation of these models is that these can be used for screening small numbers of compounds.     

Autologous melanocyte–keratinocyte suspension in the treatment of vitiligo

Background In stable vitiligo, several techniques of autologous transplantation of melanocytes are used. Autologous melanocyte transplantation of non‐cultured melanocytes is one of those techniques with variable reported outcomes. Objective The objective of this study was to evaluate the response to autologous melanocyte–keratinocytes suspension transplantation in cases of stable vitiligo. Methods A total of 25 cases of vitiligo were treated by autologous melanocyte–keratinocytes suspension transplantation. After 6–17 months, patients’ response was evaluated according to the extent of pigmentation (excellent 90–100%, good 50–89%, fair 20–49% and poor response <20%). Results Of the 25 patients treated, 22 continued the follow‐up period. Five (23%) patients showed excellent response, 7 (32%) good, 6 (27%) fair and 4(18%) showed poor response. Conclusion Unlike transplantation of cultured melanocytes, which requires experience in culture technique, autologous melanocyte–keratinocytes suspension transplantation is an easy economic technique, which may be used in resistant areas of stable vitiligo.     

Vitiligo melanocytes in long-term culture show normal constitutive and cytokine-induced expression of intercellular adhesion molecule-1 and major histocompatibility complex class I and class II molecules

The aetiology of vitiligo remains unclear. An autoimmune involvement has been suggested and, in this study, we examine whether melanocytes cultured from unaffected regions of the skin of vitiligo patients are more susceptible to immune attack by investigating constitutive and cytokine-stimulated expression of intercellular adhesion molecule-1 (ICAM-1) (under three media variants) and major histocompatibility complex (MHC) class I and class II (under one medium). Both normal and vitiligo melanocytes had similarly low constitutive expression of ICAM-1 and MHC class II molecules, whereas > 95% of cells had high constitutive expression of MHC class I. Normal and vitiligo melanocytes showed similar and significant increases in the expression of all three immune-related molecules in response to the cytokine, interferon-gamma. The expression of ICAM-1 was also similarly increased by the cytokine, tumour necrosis factor-alpha in both cells. Additionally, it was noted that, once the melanocyte cultures were established under experimental conditions, the rate of proliferation of vitiligo melanocytes did not differ significantly from that of normal melanocytes. In conclusion, we suggest that vitiligo melanocytes, once in culture, do not have intrinsic differences from normal melanocytes with respect to the expression of immune-related molecules.     

白癜风表皮黑素细胞超微结构及小眼畸形相关转录因子转录调控研究

目的 研究白癜风黑素细胞超微结构和小眼畸形相关转录因子 （MITF）及其转录调控的酪氨酸酶相关蛋白（TRP）与白癜风临床类型与病程的相关性。方法 选择不同病程的寻常型白癜风（VV）12例和节段型白癜风（SV）8例,分别取白斑区、白斑边缘正常肤色区和远离白斑正常肤色区的表皮片,经组织学确定其表皮的完整性。透射电镜观察10例患者（VV 6例,SV 4例）不同区表皮黑素细胞的超微结构特点。对所有20例远离白斑正常肤色区的表皮片黑素细胞进行培养,应用免疫印迹方法检测 MITF及其转录调控的酪氨酸酶（TYR）、酪氨酸酶相关蛋白1（TYRP1）和酪氨酸酶相关蛋白2（TYRP2）的表达水平。结果 白癜风表皮黑素细胞超微结构病理改变：10例中7例白斑区表皮内未见黑素细胞,1例短病程和2例长病程VV分别可见少量黑素体显著减少或缺失的黑素细胞;白斑边缘正常肤色区,6例VV中,3例病程小于15个月者可见黑素细胞超微结构异常,而4例SV中仅1例异常;远离白斑正常肤色区,10例黑素细胞超微结构均正常。白癜风表皮黑素细胞MITF及其转录调控TRP的表达：VV的MITF表达下调与TYR、TYRP1、TYRP2的表达下调一致;SV存在MITF显著表达下调,而TYR、TYRP1、TYRP2几均正常表达。结论 VV和SV可能存在不同的表皮黑素细胞超微结构病理改变和MITF转录调控机制。     

A study of autologous melanocyte transfer in treatment of stable vitiligo

BACKGROUND: Replenishing melanocytes selectively in vitiliginous macules by autologous melanocytes is a promising treatment. With expertise in culturing melanocytes, it has now become possible to treat larger recipient areas with smaller skin samples. AIM: To study the extent of repigmentation after autologous melanocyte transplantation in patients with stable vitiligo. METHODS: The melanocytes were harvested as an autologous melanocyte rich cell suspension from a donor split thickness graft. Melanocyte culture was performed in selected cases where the melanocyte cell count was insufficient to meet the requirement of the recipient area. These cells were then transplanted to the recipient area that had been superficially dermabraded. RESULTS: An excellent response was seen in 52.17% cases with the autologous melanocyte rich cell suspension (AMRCS) technique and in 50% with the melanocyte culture (MC) technique. CONCLUSION: Autologous melanocyte transplantation can be an effective form of surgical treatment in stable but recalcitrant lesions of vitiligo.