Immunoreactivity of α-melanocyte-stimulating hormone, adrenocorticotrophic hormone and β-endorphin in cutaneous malignant melanoma and benign melanocytic naevi

Melanocyte-stimulating hormone (MSH) has been reported to enhance the experimental metastatic behaviour of melanoma cells in the mouse model. α-MSH production and MSH receptor (melanocortin 1 receptor gene) expression have been detected in cultured normal human melanocytes and metastasized melanomas. The exact role of MSH in the metastatic behaviour of human melanoma cells is, however, not yet known. To clarify a possible role of proopiomelanocortin (POMC)-derived peptides, including α-MSH, in melanoma development and progression, we analysed immunohistochemically the localization of α-MSH, adrenocorticotrophic hormone (ACTH) and β-endorphin in various kinds of benign pigmented naevocytic lesions and malignant melanomas.   Three of 21 samples of common and dysplastic naevi showed detectable α-MSH staining in naevus cells, and five and six of 15 samples were weakly positive for ACTH and β-endorphin staining, respectively. In melanoma samples, 24 of 45, 23 of 39 and 30 of 42 samples showed positive staining with α-MSH, ACTH and β-endorphin antibodies, respectively. Furthermore, staining for all three antibodies was noted to be more intense and diffuse in samples of nodular melanoma, vertically growing acral lentiginous melanoma and superficial spreading melanoma as well as metastatic lesions compared with those of naevi. Although it is yet to be determined whether or not this strong staining for POMC-derived peptides in advanced melanoma cells indicates a role of autocrine or paracrine regulation, our results suggest a possible involvement of POMC gene products in melanoma progression.     

Thy-1+ dendritic cells in murine epidermis are bone marrow-derived

Thy-1+, Ly-5+ dendritic cells have recently been described as a resident cell population in murine epidermis, but their ontogeny and function are unknown. We therefore investigated the origin and turnover of epidermal Thy-1+ cells utilizing chimeric mice. Lethally x-irradiated AKR/J (Thy-1.1+) and AKR/Cum (Thy-1.2+) mice were reconstituted with allogeneic bone marrow cells with or without thymocytes from congenic AKR/Cum or AKR/J mice, respectively. The density of residual indigenous Thy-1.1+ cells in AKR/J chimeras and Thy-1.2+ cells in AKR/Cum chimeras was substantially reduced following x-irradiation, as determined by immunofluorescence staining of epidermal sheets. Epidermal repopulation by allogeneic Thy-1+ dendritic epidermal cells was first observed at 5 weeks in AKR/J chimeras and at 7 weeks in AKR/Cum chimeras and progressed slowly. Repopulation was not enhanced by increasing the number of allogeneic bone marrow cells injected from 2 X 10(7) to 10(8) cells or by the addition of 8 X 10(7) allogeneic thymocytes to the donor inoculate. Epidermal repopulation by allogeneic Thy-1.2+ cells was not seen in AKR/J mice reconstituted with syngeneic bone marrow cells and allogeneic Thy-1.2+ AKR/Cum thymocytes. Taken together, these results indicate that Thy-1+ dendritic epidermal cells are derived from the bone marrow and suggest that they are not related to conventional peripheral T-lymphocytes.     

双链DNA抗原冲激导致髓源性树突细胞免疫表型变化

目的 探索外源性双链DNA物质对小鼠髓源性树突细胞（DC）免疫表型的影响。方法 采用免疫磁珠法分离C57小鼠骨髓lin-CD117+ 干细胞,用多种细胞因子诱导后其增殖并发育成不同成熟阶段的DC。提取马疫锥虫动基体DNA（kDNA）,对上述DC进行冲激。采用流式细胞法和激光共聚焦显微镜检测DC免疫表型和形态学变化。结果 冲激前,未成熟、半成熟和成熟DC的MHCⅡ阳性率依次为11.42% ± 2.56%、27.08% ± 5.29%与44.63% ± 10.37%,CD80阳性率为8.54% ± 2.01%、31.35% ± 6.40%与52.96% ± 10.34%,CD86阳性率为10.22% ± 3.47%、32.15% ± 6.83%与64.72% ± 9.68%。冲激后,这三组DC的MHCⅡ阳性率分别上升15.63%、9.66%、4.12%,与冲激前比较,t值分别为6.21、4.35与2.82,P值均 < 0.05;CD80阳性率上升9.63%、7.09%与4.09%,CD86阳性率上升13.16%、9.75%与3.10%,升高幅度皆为未成熟DC > 半成熟DC > 成熟DC。结论 双链DNA抗原可促进髓源性DC表达成熟免疫表型,且成熟程度越低的DC受影响越显著。     

Identification of genes specifically regulated in human melanoma cells

In order to improve the characterization of human malignant melanoma cells and their variant gene expression in vitro, a search for specifically regulated genes was performed. Four melanoma cell lines (M5, MEWO, IGR39, SKMEL13) and newly cultured normal human melanocytes were included in a comparative hybridization (differential screening) of a human melanoma cDNA-library. Six cDNAs were isolated showing a stronger expression (four genes) or a weaker expression (two genes) in melanoma cells than in normal human melanocytes. Quantification of the expression patterns of the two repressed genes in Northern blots revealed general expression in all melanocyte cultures examined, no expression in three cell lines (M5, IGR39, SKMEL13) and weak expression in MEWO. The four induced genes were found to be only weakly expressed in normal human melanocytes, but showed an elevated expression in all of the four melanoma cell lines tested. Thus, using the technique of differential screening, consistent gene regulation at the messenger RNA level was identified, which distinguishes the four melanoma cell lines tested from normal melanocytes. We conclude from the expression patterns that specific gene regulation in melanoma cells in vitro is characterized both by strong repression of some melanocyte genes, as well as by the induction of other genes, but there was no indication of new expression of genes specific for melanoma cells. Because of the uniform induction or repression in different melanoma cell lines, it is conceivable that the cloned genes may be involved in the malignant transformation of melanocytic cells.     

Effect of pimecrolimus vs. corticosteroids on murine bone marrow-derived dendritic cell differentiation, maturation and function

Abstract:  Pimecrolimus (SDZ ASM981) is a non-steroid member of calcineurin inhibitors recently developed for the treatment of inflammatory skin diseases. In this study, we compared the effect of pimecrolimus and corticosteroids on the differentiation, maturation and function of murine bone marrow-derived dendritic cells (BM-DC). We added pimecrolimus at concentrations of 5–500 ng/ml or 0.5 ng/ml mometasone furoate at different timepoints to the BM-DC culture and checked (i) the number of matured cells, (ii) the expression of activation markers, (iii) the release of cytokines and (iv) the stimulatory capacity of the resulting BM-DC in vivo . Even at the highest concentration, pimecrolimus treatment resulted in only modest effects. In the pimecrolimus-treated culture, we observed a decrease in the numbers of matured cells but no significant effects on the expression of activation markers. The release of some inflammatory cytokines was reduced, but the stimulatory capacity in vivo was not affected. In contrast, mometasone furoate has pronounced effects on BM-DC at a concentration ten to 1000 times lower than those used with pimecrolimus. Furthermore, topical treatment of mice with clobetasole cream 0.05% resulted in almost complete depletion of splenic DC and a severe hyposplenia, while high-dose oral pimecrolimus treatment did not show any effects on the spleen or on splenic DC. These results support that pimecrolimus, unlike corticosteroids, has little effects on dendritic cells. To the best of our knowledge, this is the first study of this type with use of BM-DC.     

CARD9基因敲除小鼠骨髓来源树突状细胞对阿萨希毛孢子菌临床株的免疫反应缺陷

目的初步探讨CARD9基因敲除小鼠骨髓来源树突状细胞(bone marrowderived dendritic cell,BMDC)对阿萨希毛孢子菌临床株(Trichosporon asahii,T.asahii)的免疫反应缺陷。方法体外培养野生型(wild type,WT)与CARD9基因敲除型(CARD9knockout,CARD9-/-)小鼠BMDC并分别与热灭活的T.asahii进行共培养,比较两者对菌体的黏附吞噬能力、表面共刺激分子的激活、细胞因子的表达以及两种小鼠感染菌株后的生存率。结果共培养24 h后,WT与CARD9-/-小鼠BMDC对T.asahii的黏附吞噬情况比较未见明显差异;经T.asahii刺激的CARD9-/-小鼠BMDC的CD80、CD86激活情况以及白细胞介素(IL)-6、肿瘤坏死因子(TNF)-α表达水平均明显低于WT小鼠BMDC(P0.05);感染T.asahii的CARD9-/-小鼠的生存率明显低于WT小鼠(P0.05)。结论 CARD9-/-小鼠BMDC对T.asahii的免疫反应缺陷主要体现在共刺激分子的激活以及促炎细胞因子的表达,但并未影响其对T.asahii的吞噬识别。     

FACS purification of bone marrow-derived epidermal populations in mice: Langerhans cells and Thy-1+ dendritic cells

A method was developed which allows for the separation and purification of Langerhans cells (LC) and Thy-1+ cells (Thy-1+dEC) from mouse epidermis. Epidermal cell (EC) suspensions were subjected to Ficoll separation, and the resulting interface EC were harvested. These EC were then "tagged" with the appropriate monoclonal antibody and sorted into positive and negative populations using the Fluorescence Activated Cell Sorter (FACS). Preparations of viable LC and Thy-1+dEC were obtained with 94-98% and 94-99% purities, respectively.     

Suprabasal expression of epidermal α2β1 and α3β1 integrins in skin treated with topical retinoic acid

In normal adult human skin, expression of epidermal integrins is confined to keratinocytes in the basal layer. However, suprabasal expression of α2, α3 and β1 integrin subunits is noted in hyperproliferative epidermis in wound repair and psoriasis. In this study, we examined the effect of topical all- trans -retinoic acid (RA), known to induce epidermal hyperplasia, on expression of integrins in human epidermis. Immunostaining of vehicle-treated skin revealed expression of α2, α3 and β1, as well as α6 and β4 integrin subunits entirely on basal keratinocytes. Topical application of RA (0.1%) for 2 weeks resulted in marked suprabasal expression of α2, α3 and β1 integrin subunits, whereas α6 and β4 staining remained on basal keratinocytes. Staining for putative ligands of α2β1 and α3β1 integrins, i.e. type IV collagen, laminin-5 and fibronectin, was not detected in the epidermal layer in RA- or vehicle-treated skin. Treatment of HaCaT keratinocytes in culture with RA (1 μmol/L) enhanced α2 and β1 mRNA abundance. Furthermore, RA slightly up-regulated the expression of α2, α3 and β1 integrin subunits on primary epidermal keratinocytes and HaCaT cells in culture with no effect on cell proliferation. These results provide evidence that RA-elicited epidermal hyperplasia is associated with aberrant suprabasal expression of α2β1 and α3β1 integrins, and that this also involves direct stimulation of keratinocyte integrin expression by RA.     

Structure-activity relationships in the murine local lymph node assay for skin sensitization: α,β-diketones

The biological activity of skin-sensitizing chemicals is related to their ability to react, either directly or after metabolic activation, with appropriate skin proteins. For direct acting electrophilic compounds, this ability can be modelled, using the RAI (relative alkylation index) approach, by a combination of electrophilicity and hydrophobicity parameters. Several structure-activity relationships based on this approach have been reported, but most of them cover guinea pig sensitization test data on what chemists would classify as relatively soft electrophilic chemicals. In the present work, an electrophilicity parameter based on Taft substituent constants is derived for hard electrophiles having a reactive carbonyl group, and is used to calculate RAI values for the analysis of sensitization test data obtained in the murine local lymph node assay (LLNA) for a series of alpha, beta-diketones. The sensitization potential of these reactive hard electrophilic carbonyl compounds in the LLNA shows a good correlation with the RAI. Overall, the findings reaffirm our view that physical organic chemistry is the key to understanding why some chemicals sensitize more strongly than others, while some do not sensitize at all, and provide further evidence of the value of the LLNA for SAR studies.     

Fibroblast expression of collagen integrin receptors α1β1 and α2β1 is not changed in systemic scleroderma

The skin of patients with systemic scleroderma (SSc) is characterized by excessive extracellular matrix deposition in the dermis. As collagens represent the major structural component, we used fluorescence-activated cell sorter analysis to study the levels of collagen receptors expressed at the surface of fibroblasts derived from involved skin areas. In contrast to previous reports, no differences in the expression of alpha1, alpha2 or beta1 integrin subunits, which constitute the major collagen receptors on fibroblasts, were detected on SSc fibroblasts as compared with normal control fibroblasts. Variation of cell culture conditions, e. g. passage number (from 2 to 10), seeding density, cell cycle or serum concentration, did not change this result. These observations indicate that any abnormal response of SSc fibroblasts to their matrix environment is not controlled at the level of receptor expression.     

Effect of PUVA on plasma and skin immunoreactive α-melanocyte stimulating hormone concentrations

Plasma alpha-melanocyte stimulating hormone (alpha-MSH) concentrations were measured in patients receiving PUVA therapy as treatment for mycosis fungoides, and PUVA or UVB as treatment for psoriasis. Skin immunoreactive alpha-MSH was also measured in those patients who received PUVA. The mean plasma and skin alpha-MSH concentrations after 2-3 weeks of PUVA were not significantly different from pre-treatment values and showed no relationship either to skin type or to the degree of tanning that occurred in response to PUVA. Plasma alpha-MSH concentrations were also unchanged after UVB. There was also no short term change in plasma alpha-MSH concentrations in patients after receiving their first treatment with PUVA. It would appear that circulating and skin alpha-MSH levels are unaffected by UV and show no causal relationship to PUVA induced pigmentation.     

The depigmenting effect of α-tocopheryl ferulate on human melanoma cells

Oral vitamin E (alpha-tocopherol, alpha-T) supplementation has been reported to improve facial hyperpigmentation. alpha-Tocopheryl ferulate (alpha-TF) is a compound of alpha-T and ferulic acid connected by an ester bond; ferulic acid is also an antioxidant, and could scavenge free radicals induced by ultraviolet (UV) radiation, and thus maintain the long-lasting antioxidative effect of alpha-T. Our aim was to see whether alpha-TF might be useful as a whitening agent and an antioxidant to improve and prevent facial hyperpigmentation following UV exposure. In this study, the inhibitory effect of alpha-TF on melanogenesis was examined biochemically using human melanoma cells in culture. The results show that alpha-TF, solubilized in ethanol or in 0.5% lecithin, inhibited melanization significantly, as did alpha-T at a concentration of 100 microg/mL, without inhibiting cell growth. This phenotypic change was associated with inhibition of tyrosinase and 5, 6-dihydroxyindole-2-carboxylic acid polymerase activities, and the degree of inhibition was dose dependent. No significant effect on DOPAchrome tautomerase activity was observed. alpha-TF did not directly inhibit tyrosinase activity of the large granule fraction extracted from human melanoma cells, and Western blotting revealed that there were no changes in protein content or in molecular size of tyrosinase, tyrosinase-related protein (TRP)-1 or TRP-2. Therefore, the inhibition of tyrosinase activity by alpha-TF might be due to effects at the post-translational level, and possibly by a secondary molecule activated by alpha-TF. These results suggest that alpha-TF is a candidate for an efficient whitening agent which suppresses melanogenesis and inhibits biological reactions induced by reactive oxygen species.     

Thy-1 antigen-bearing dendritic cells in murine epidermis are derived from bone marrow precursors

Thy-1 antigen is expressed on a dendritic subpopulation of cells in murine epidermis. Numbering between 200 and 500/mm2 surface area in abdominal skin, they are distinct from the dendritic Langerhans cells (LCs) and melanocytes. Since immigrant lymphoid cells as well as constitutive cells in various organs have been demonstrated to be Thy-1+, their origin and function are not certain. To assess these issues, two experimental protocols were established. First, grafts of whole skin from AKR mice were placed orthotopically on (AKD2)F1 recipients. Immigration of recipient-derived cells into graft epidermis was assessed histologically by fluorescence microscopy employing monoclonal anti-Thy-1.2 and anti-I-Ad antibodies. Second, bone marrow chimeras were established in AKR recipients after lethal irradiation and reconstitution with cells from (AKD2)F1 donors. In the first protocol, dendritic I-Ad+ LCs of donor origin infiltrated each graft to normal densities within 2 weeks. Thy-1.2+ cells also immigrated into the same grafts, but at much slower rates. In the second protocol, bone marrow-derived Thy-1.2+ cells populated normal skin epidermis slowly over several months, with densities reaching 70/mm2. We conclude that some, if not all, Thy-1+ cells in normal murine epidermis are derived from bone marrow precursors, that their infiltration rates differ substantially from those of LCs, and that those factors which govern immigration rates into adult skin derive from the skin itself rather than from the systemic availability of their precursors. We suggest that the function of Thy-1+ epidermal cells will therefore reside among those usually ascribed to recirculating hematogenous cells, including the possibility that they may down-regulate immunizing signals that emerge from skin.     

SOCS调控树突状细胞在炎症性皮肤病中的作用机制研究进展

细胞因子信号抑制因子基因可通过对树突状细胞成熟分化的调控,影响免疫应答的平衡稳定,在炎症性疾病的发生、发展中具有重要影响。本文就细胞因子信号抑制因子基因调控树突状细胞在银屑病、类风湿性关节炎、系统性红斑狼疮、真菌感染等炎症性疾病中的作用机制研究进展进行综述。     

Growth and pigmentation in genetically related Cloudman S91 melanoma cell lines treated with 3-isobutyl-1-methyl-xanthine and β-melanocyte-stimulating hormone

Abstract 4 clonal sublines of Cloudman S91 melanoma cells, S91/mel, S91/13, S91/6 and S91/amel, were evaluated for changes in growth, pigment content and plating efficiency during and after treatment with a cyclic-AMP phosphodiesterase inhibitor-melanin-stimulating agent, 3-isobutyl-l-methyl-xanthine (IBMX) plus β-melanocyte stimulating hormone (β-MSH) or IBMX alone. After combined treatment, increases in melanin content on day 3 were 48, 27, 11, and 2 pg/cell in the four cell lines respectively. In each case IBMX alone was less effective than IBMX plus β-MSH. Doubling time increased and plating efficiency decreased with increased melanization. The increases in doubling time and decreases in plating efficiency were cell line dependent. The greatest rate of increase in doubling time and decrease in plating efficiency as a function of melanin content were seen in S91/amel, which produced the least pigment. The lowest rates of increase/decrease were seen in S91/mel, which produced the most pigment. Melanin pigment induced in the cells was classified as etimelanin by EPR determination. The differential response to induction of pigmentation makes these cell lines suitable models for comparative studies on the role of melanin in pigment cell biology.