Expression of BMI-1 in normal skin and inflammatory and neoplastic skin lesions

BACKGROUND: BMI-1 is involved in the maintenance of stem cells and functions as an oncogene in both lymphomas and solid carcinomas, acting by downregulation of p16ink4a. We have investigated the expression profile of BMI-1 in normal and inflamed skin as well as in basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs). METHODS: BMI-1 expression was determined by immunohistochemistry and immunofluorescence, and evaluated semiquantitatively. RESULTS: BMI-1 was weakly expressed in nuclei of basal and sometimes suprabasal keratinocytes, in basal cells of sebaceous glands, weakly to moderately in the bulge area and the external root sheath of hair follicles, and strongly in sweat glands. Whereas BCCs showed strong and diffuse BMI-1 expression, SCCs expressed BMI-1 heterogeneously. Strong cytoplasmic expression of BMI-1 was found in dividing cells. CONCLUSIONS: BMI-1 expression marks stem cells within the hair follicle. As BMI-1 was also found in suprabasal keratinocytes and a variety of specialized cells, the distribution of BMI-1 only partly reflects the known distribution of stem cell compartments. BMI-1 is strongly overexpressed in BCCs, tumors linked to dysregulation of the sonic hedgehog pathway, which has been shown to upregulate BMI-1, suggesting a contribution of the BMI-1 oncogene in their pathogenesis.     

B1 and B2 kinin receptor participation in hyperproliferative and inflammatory skin processes in mice

Background

Kinins are released during dermal injury and inflammation and seem to contribute to the pathogenesis of cutaneous diseases.

Objective

Participation of kinins in skin inflammatory process was evaluated using knockout mice and non-peptide kinin receptor antagonists.

Methods

Chronic skin inflammation was induced by multiple applications of TPA in mice ear.

Results

The B₂ knockout mice (B₂^−/−) showed a significant increase of ear weight (23 ± 10%) and epidermal cellular hyperproliferation and acanthosis formation upon histological analysis when compared with wildtype mice. Also, evaluation of PCNA levels by Western blot and immunohistochemistry confirmed the increase in the epidermis hyperproliferation in the ear skin of B₂^−/− mice. In contrast, no modification in these parameters was detected in B₁ knockout mice (B₁^−/−). However, mice lacking both kinin receptors (B₁B₂^−/−) presented a considerable reduction of epidermis thickness and in PCNA levels. Following the establishment of skin inflammation (5th day of TPA application) treatment with the non-peptide antagonists SSR 240612 (B₁ receptor antagonist), FR 173657 (B₂ receptor antagonist), or SSR 240612 plus FR 173657 topically applied, caused a significant inhibition of ear weight (20 ± 5%, 34 ± 4% and 32 ± 6%, respectively). In the histological analysis, the antagonists produced a reduction in epidermal hyperplasia and acanthosis formation; but the treatment with a combination of the two antagonists did not increase efficacy.

Conclusion

Kinin receptors seem to be involved in the control of the keratinocyte hyperproliferative process, and non-peptide kinin receptor antagonists may be useful tools in the treatment of hyperproliferative skin disorders.     

Expression and function of the mannose receptor CD206 on epidermal dendritic cells in inflammatory skin diseases

The capability to take up mannosylated protein antigens is important for the biologic function of dendritic cells, as many glycoproteins derived from bacteria and fungi, e.g., Malassezia furfur, are mannosylated. The expression of the mannose receptor CD206 has been regarded a differentiation hallmark of immature dendritic cells, whereas monocytes and mature dendritic cells as well as epidermal Langerhans cells do not express CD206. This study describes some epidermal dendritic cells that may express CD206 under inflammatory skin conditions: Immunohistochemical and flow cytometric analysis with the CD206-specific D547 antibody confirmed that Langerhans cells from normal human skin do not express CD206. Epidermal cell suspensions from atopic dermatitis and psoriasis revealed two distinct subsets of epidermal dendritic cells: a CD1a(+++)/CD206(-) cell population (i.e., Langerhans cells) and a CD1a(+)/CD206(++) cell population, corresponding to the previously described inflammatory dendritic epidermal cells. CD206-mediated endocytosis, assessed by dextran-fluorescein isothiocyanate uptake, was demonstrated in inflammatory dendritic epidermal cells but not in Langerhans cells. CD206-independent uptake of the fluorescent dye Lucifer yellow, a pinocytosis marker, was demonstrated in both Langerhans cells and inflammatory dendritic epidermal cells. Electron microscopic examination, known to distinguish Langerhans cells from inflammatory dendritic epidermal cells by their Birbeck granules, revealed Langerhans cells with Birbeck granules and inflammatory dendritic epidermal cells without Birbeck granules. Inflammatory dendritic epidermal cells exhibited numerous coated pits and vesicles, the latter fusing with large endosome-like structures, thus suggesting a high endocytotic activity. Immunogold staining with D547 monoclonal antibody confirmed that inflammatory dendritic epidermal cells were positive for CD206. In conclusion, inflammatory dendritic epidermal cells but not Langerhans cells are expressing CD206 in situ and use it for receptor-mediated endocytosis.     

Expression of allograft inflammatory factor-1 in inflammatory skin disorders

Allograft inflammatory factor-1 (AIF-1) is an evolutionarily conserved, inflammatory protein produced by activated macrophages during chronic transplant rejection and in inflammatory brain lesions. Since T-cell-mediated inflammation is common to various dermatoses and nothing is known about AIF-1 in skin, we studied its protein expression at the tissue level and regulation in monocytic cell lines by various agents. Using immunohistochemistry, we found that AIF-1 is expressed at low levels in normal skin, but is highly upregulated in various inflammatory skin disorders, such as psoriasis, lichen planus, graft-versus-host disease and mycosis fungoides. The main cell types expressing AIF-1 in affected skin are macrophages and Langerhans' cells. We also show by real-time PCR that AIF-1 mRNA levels in monocytic THP-1 and U937 cell lines are significantly upregulated by retinoic acid as well as a number of cytokines. We conclude that AIF-1 may mediate survival and pro-inflammatory properties of macrophages in skin diseases.     

Dermcidin is constitutively produced by eccrine sweat glands and is not induced in epidermal cells under inflammatory skin conditions

BACKGROUND: Antimicrobial peptides (AMPs) are important effector molecules of innate immunity, protecting epithelial surfaces of multicellular organisms. In human skin two classes of AMPs-the beta-defensins and the cathelicidins-are produced by keratinocytes primarily under inflammatory conditions. In contrast, dermcidin (DCD), a recently discovered AMP with broad-spectrum activity, is expressed in eccrine sweat glands and transported via sweat to the epidermal surface. OBJECTIVES: To investigate whether DCD expression is induced under inflammatory conditions in epidermal keratinocytes. METHODS: Lesional skin of the inflammatory skin diseases atopic dermatitis, psoriasis and lichen planus was analysed by immunohistochemistry using a polyclonal anti-DCD antiserum. We also examined whether DCD RNA expression is induced in cultured human keratinocytes, fibroblasts, melanocytes and melanoma cells. RESULTS: Whereas DCD was constitutively expressed in eccrine sweat glands of all skin biopsies, we found that, independent of the type of the inflammatory skin lesion, DCD protein expression was not induced in human epidermal keratinocytes. In contrast, beta-defensin 2 was expressed in epidermal keratinocytes of inflammatory human skin, but not in keratinocytes of healthy human skin. Upon stimulation of the cultured cells with 12-O-tetradecanoyl-phorbol-13-acetate, tumour necrosis factor-alpha, lipopolysaccharide or H2O2, DCD mRNA expression was not detected in primary keratinocytes, fibroblasts and melanocytes, but was detected in MeWo and SKMEL28 melanoma cells. CONCLUSIONS: These results indicate that, unlike human cathelicidins and beta-defensins which are inducible peptides that primarily function in response to injury and inflammation, DCD is exclusively part of the constitutive innate defence of human skin. By modulating surface colonization, DCD may help to prevent local and systemic invasion of pathogens.     

Expression of T-cell receptor antigens in mycosis fungoides and inflammatory skin lesions

Using immunohistologic methods, we studied the expression of the T-cell receptor (TCR)-associated antigens CD3, TCR-beta, and TCR-delta by cutaneous T cells in mycosis fungoides (MF) (36 patients) and a variety of inflammatory diseases (16 patients). Most T cells in the inflammatory diseases and patch/plaque mycosis fungoides expressed the immunophenotype characteristic of the vast majority of mature peripheral T cells: CD3+ TCR-beta+ TCR-delta-. In contrast, abnormal CD3/TCR-beta antigen expression was seen in 3 of 6 cases (50%) of tumor stage mycosis fungoides. Furthermore, we were able to document its evolution from the normal pattern present in earlier patch/plaque lesions of the two cases in which serial biopsies were available for study. Divergence of epidermal versus dermal CD3/TCR-beta antigen expression was seen in 2 of 34 (6%) of biopsies of patch/plaque mycosis fungoides but not in inflammatory controls. The TCR-delta+ cells were generally rare regardless of diagnosis. We conclude that inflammatory skin diseases and most patch/plaque mycosis fungoides are typically composed of T lymphocytes that resemble mature peripheral T cells in regard to their expression of TCR-associated antigens. In contrast, aberrant patterns of TCR-associated antigen expression can be seen in tumor stage MF, and, more rarely in patch/plaque MF.     

Atopic dermatitis: immunophenotyping of inflammatory cells in skin lesions

BACKGROUND: The ever increasing incidence of atopic dermatitis (AD) has stimulated many researchers to use various diagnostic procedures to obtain new data to help elucidate the pathogenesis of the disease. AIMS: To perform cell immunophenotyping and to analyze the presence of inflammatory cell-surface markers in the biopsies of skin lesions from 15 AD patients and five healthy subjects. METHODS: Immunohistochemical analysis was performed in a group of AD patients and compared with that in a control group of healthy subjects. Avidin-biotin immunoperoxidase staining of paraffin-embedded, 4 microm skin sections, with semiquantitative counting of cells labeled with anti-CD3, anti-CD8, anti-CD20, anti-HLA-DR (HLA, human leukocyte antigen), and anti-immunoglobulin E (anti-IgE) primary antibodies, was used. RESULTS: The results of AD skin analysis showed a greater infiltration of CD3+ lymphocytes, especially of CD4+ subtype, compared with CD8+ lymphocytes. AD skin biopsy specimens also showed a higher intraepidermal HLA-DR+ Langerhans' cell count, the presence of HLA-DR on lymphocytes in the dermis, and higher intraepidermal expression of IgE+ cells compared with healthy controls. CONCLUSIONS: A statistically significant difference (P < 0.05) was found between the two groups for intradermal and intraepidermal CD3, CD4, and HLA-DR, intradermal CD8, and intraepidermal IgE+ cells. Immunophenotyping was found to be a useful diagnostic method in AD patients.     

Suitability of macrophage inflammatory protein-1beta production by THP-1 cells in differentiating skin sensitizers from irritant chemicals

Background:  Worldwide restrictions in animal use for research have driven efforts to develop alternative methods.
Objective:  The study aimed to test the efficacy of the macrophage inflammatory protein-1β (MIP-1β) assay for testing chemicals' skin-sensitizing capacity.
Methods:  The assay was performed using 9 chemicals judged to be sensitizing and 7 non-sensitizing by the standard in vivo assays. THP-1 cells were cultured in the presence or absence of 4 doses, 0.01x, 0.1x, 0.5x, or 1x IC₅₀ (50% inhibitory concentration for THP-1 cell proliferation) of these chemicals for 24 hr, and the MIP-1β level in the supernatants was determined. Skin sensitization by the test chemicals was determined by MIP-1β production rates. The MIP-1β production rate was expressed as the relative increase in MIP-1β production in response to chemical treatment compared with vehicle treatment.
Results and Conclusion:  When the threshold MIP-1β production rate used was 100% or 105% of dimethyl sulfoxide, all the sensitizing chemicals tested (dinitrochlorobenzene, hexyl cinnamic aldehyde, eugenol, hydroquinone, dinitrofluorobenzene, benzocaine, nickel, chromium, and 5-chloro-2-methyl-4-isothiazolin-3-one) were positive, and all the non-sensitizing chemicals (methyl salicylate, benzalkonium chloride, lactic acid, isopropanol, and salicylic acid), with the exception of sodium lauryl sulfate, were negative for MIP-1β production. These results indicate that MIP-1β could be a biomarker for classification of chemicals as sensitizers or non-sensitizers.     

Polyamine-dependent post-translational modification of proteins in differentiating mouse epidermal cells

In order to get a better understanding of the role played by polyamines in calcium-induced epidermal cell differentiation, the time course of their metabolism was investigated. Results demonstrate that differentiating epidermal cells are characterized by time-dependent changes in polyamine concentrations. An early polyamine catabolic phase, characterized by increased total putrescine concentration and drastic reduction of both spermidine and spermine levels, is followed by active spermidine biosynthesis. The differences in putrescine and, in particular, spermidine metabolism are reflected in a time-dependent modulation of protein-bound polyamine derivatives. In fact, upon addition of calcium to the culture medium, hypusine N epsilon-(4-amino-2-hydroxybutyllysine) is rapidly reduced to undetectable levels. The very low hypusine level is paralleled by an increase in gamma-glutamyl putrescine derivatives and followed by a large increase in gamma-glutamyl spermidine derivatives; in addition, there is a remarkable concomitant biosynthesis of transglutaminase-catalyzed mono and bis gamma-glutamyl spermidine derivatives and epsilon(gamma-glutamyl)lysine cross-links. The effect of TPA and RA on hypusine formation is also reported.     

Ovariectomy is sufficient to accelerate spontaneous skin ageing and to stimulate ultraviolet irradiation-induced photoageing of murine skin

Background: Wrinkling and sagging of the skin during photoageing is physiologically associated with diminished elasticity, which can be attributed to increased fibroblast-derived elastase activity. This degrades the dermal elastic fibres needed to maintain the three-dimensional structure of the skin. We previously reported that ovariectomy accelerates ultraviolet (UV)B-induced wrinkle formation in rat hind limb skin by altering the three-dimensional structure of elastic fibres. OBJECTIVES: In this study, we used hairless mice to assess the effects of ovariectomy with or without chronic UVA or UVB radiation on sagging and wrinkling of skin, on the elasticity of skin, as well as on matrix metalloproteinase activities in the skin. METHODS: Ovariectomies or sham operations were performed on 6-week-old female ICR/HR hairless mice. RESULTS: Even in the ovariectomy group without UV irradiation, the skin elasticity was significantly decreased during the 3-13 weeks after ovariectomy, which was accompanied by a significant increase in elastase activity in the skin. After UVA or UVB irradiation, skin elasticity was significantly decreased to a greater extent in the ovariectomy group than in the sham operation group, and this was accompanied by a reciprocal increase in elastase activity but not in the activities of collagenases I or IV in the skin. Consistent with the decreased skin elasticity, UVA irradiation for 12 weeks elicited more marked sagging in the ovariectomy group than in the sham operation group. UVB irradiation for 12 weeks also induced more marked wrinkle formation in the ovariectomy group than in the sham operation group. CONCLUSIONS: These results suggest that ovariectomy alone is sufficient to accelerate skin ageing and to increase UV sensitivity, which results in the further deterioration of the skin and photoageing, and may account for the accelerated skin ageing seen in postmenopausal women.     

Expression of Thy-1 antigen by murine epidermal cells

We report on the occurrence of a cell population within the murine epidermis which, by both morphologic and surface property criteria, is distinct from all other epidermal cell types known so far. These previously unrecognized cells are evenly distributed within the epidermis, display a primarily dendritic shape, exhibit a lobulated nucleus, contain large amounts of vimentin type intermediate-sized filaments, but lack desmosomes, melanosomes, Merkel cell granules, and Birbeck granules. As opposed to melanocytes, these cells fail to display tyrosinase activity. Surface marker analysis reveals these cells to uniformly express the Thy-1 antigen and to lack I-A and I-E/C antigen specificities. A major portion of these Thy-1-bearing cells are reactive with a monoclonal antibody to the Ly-5 determinant whereas attempts to demonstrate Lyt-1,2,3 antigens consistently yield negative results. These findings strongly suggest that Thy-1+ epidermal cells originate from the bone marrow; however, their precise relationship to distinct members of the hemopoietic differentiation pathway remains to be established.     

玫瑰糠疹皮损血管内皮细胞细胞间黏附分子-1表达及其超微结构研究

目的:探索玫瑰糠疹的发病机制。方法:采用免疫组化法检测玫瑰糠疹皮损血管内皮细胞的细胞间黏附分子(ICAM)-1的表达,通过透射电镜观察玫瑰糠疹皮损血管内皮细胞超微结构的改变。结果:血管内皮细胞ICAM-1的表达皮损组均高于正常皮肤组及玫瑰糠疹皮损周围皮肤组,而且玫瑰糠疹皮损周围皮肤组高于正常皮肤组,在统计学上差异均有显著性(P<0.05)。5/8标本中可见真皮血管内皮细胞连接处间隙扩大,紧密连接消失或破坏。结论:ICAM-1在玫瑰糠疹皮损血管内皮细胞的高表达及玫瑰糠疹真皮血管内皮细胞超微结构的改变,可能与玫瑰糠疹部分皮损内可见数量不等的血管外红细胞有关。     

真菌荧光染色法检测健康皮肤和炎症性皮损的真菌定植

目的:利用荧光染色法检测健康皮肤和炎症性皮损的真菌阳性率及定植数量。方法:门诊收集正常皮肤及银屑病、特应性皮炎、湿疹和玫瑰糠疹患者,采集每位患者和健康对照组皮脂溢出部位(面部、胸部和背部)、干燥部位(双上肢)、潮湿部位(腹股沟、腘窝、肘窝)、头皮及足底部位的皮损皮屑,利用真菌荧光染色试剂快速染色,随机选择30个视野计数真菌(孢子)数量,数据使用R 3.5.3软件进行统计学分析。结果:共收集457例患者和888名健康对照,健康对照头皮和皮脂溢出部位的真菌检出率高分别为95%和96%,足底真菌检出率最低(44.29%)。湿疹组、银屑病组和玫瑰糠疹组皮脂溢出部位的真菌检出率分别为77.05%、66.67%、91.42%高于相应的干燥部位58.93%、47.62%、57.89%。在头皮、皮脂溢出部位、干燥部位,疾病组真菌检出率低于健康组,差异有统计学意义(均P<0.05)。健康组4个部位真菌分布的数量不同且差异有统计学意义(P<0.05)。皮脂溢出部位中,18～44岁年龄组孢子数量最多。结论:健康皮肤不同部位的真菌阳性率和孢子数量不同。特应性皮炎、湿疹、银屑病和玫瑰糠疹的皮损部位真...     

Thy-1+ epidermal cells are not demonstrable in rat and human skin

Recent studies have revealed the presence of a population of Thy-1+ epidermal cells in murine epidermis. These experiments were designed to determine whether analogous Thy-1+ cell populations occur in rat and human epidermis. Thy-1+ cells could not be demonstrated in epidermal sheets derived from rat and human skin or in epidermal cell lines, KB and A431. To date, Thy-1+ epidermal cells have been observed only in murine epithelia. The cellular equivalent of the murine Thy-1+ cell in other species remains elusive.     

Suprabasal induction of ornithine decarboxylase in adult mouse skin is sufficient to activate keratinocytes

To study the effects of de novo induction of ornithine decarboxylase (ODC) activity in adult, quiescent skin, we generated transgenic mice in which the suprabasal expression of an inducible form of the ODC protein fused to a modified estrogen receptor ligand-binding domain (ODCER) is driven by an involucrin promoter. After topical treatment with the inducing agent 4-hydroxytamoxifen (4OHT), ODC activity and putrescine levels were dramatically increased in the epidermis but not in the dermis of transgenic mice. 4OHT treatment stimulated both proliferation as measured by bromodeoxyuridine incorporation in basal epidermal cells and differentiation shown by increased expression of differentiation markers. Furthermore, induction of ODC activity did not rescue primary epidermal keratinocyte cultures isolated from ODCER2 mice from a calcium-triggered DNA synthesis block, as measured by [3H]thymidine incorporation. In vivo induction of epidermal ODC enzyme activity significantly stimulated the vascularization of ODCER transgenic skin. Increased expression of interleukin-1beta and keratin 6, markers of keratinocyte activation seen in wound healing, was also observed in 4OHT-treated transgenic skin. These results suggest that de novo suprabasal induction of ODC activity in adult mouse skin activates keratinocytes and stimulates vascularization in the dermal layer in a manner similar to skin undergoing wound healing.     

Reappraisal of in situ immunophenotypic analysis of psoriasis skin: interaction of activated HLA-DR+ immunocompetent cells and endothelial cells is a major feature of psoriatic lesions

Psoriasis is an inflammatory skin disease of unknown aetiology. Many observations indicate that T cells play an important role in the pathogenesis of the disease. Upregulation of MHC class-II molecules on immunocompetent cells, endothelial cells and keratinocytes on lesional psoriatic skin has been regarded as a hallmark of the disease. However, there is some controversy in the literature regarding the cell types expressing class-II molecules and there is limited information about the presence of immune cells other than T cells and antigen presenting cells in the cellular infiltrates of psoriatic skin. We therefore reinvestigated the subject using immunocytochemical single and multiple staining techniques. In agreement with earlier reports, our studies showed that the cellular infiltrates in lesional skin consist largely of HLA-DR⁺/IL-2R⁺ T cells, HLA-DR⁺/CD1a⁺ Langerhans cells, and HLA-DR⁺/CD68⁺ macrophages. We found increased HLA-DR expression mostly on immunocompetent cells and endothelial cells, but no prominent HLA-DR expression on keratinocytes in lesional psoriatic skin. Upregulation of HLA-DR on endothelial cells and in mononuclear infiltrates was also evident in the non-lesional skin of psoriatic patients as compared with normal controls. B cells and natural killer cells were also found in the cellular infiltrates in lesional psoriatic skin. In spite of the presence of a large amount of activated T cells in the epidermis, we found that HLA-DR expression on keratinocytes was not a major feature of psoriatic skin.