Topical tretinoin replenishes CD1a-positive epidermal Langerhans cells in chronically photodamaged human skin

Excessive exposure to sunlight results in drastic degradative changes in the constitutive cells of the epidermis. Among these is a profound abrogation of Langerhans cell number and function, leading to compromised immunologic competency. Retinoids have recently been shown to restore immunologic function in the setting of iatrogenic immunosuppresion. We asked whether topical tretinoin would reverse Langerhans cell depletion in chronically photodamaged skin.
We examined the skin of 8 volunteers immunohistochemically before and after 6 months of daily applications of tretinoin. At baseline, there was a profound depletion of CD1a-positive Langerhans cells in the interfollicular epidermis, but not in the adjoining follicular epithelium. After tretinoin, all patients demonstrated replenishment of interfollicular epidermis by CD1a-positive Langerhans cells. This was associated with induction of HLA-DR expression on infundibular keratinocytes, as well as the appearance of CD1a dendritic cells in the papillary dermis. Thus, the enhancement of epidermal immunity in photodamaged skin may reflect restoration of antigen-presenting Langerhans cells. The source of this renewed dendritic cell population is likely to be follicular infundibulum and the papillary dermis.     

Characterization of human skin-derived CD1a-positive lymph cells

Abstract The phenotype and function of CD1a⁺ lymph cells is of considerable interest. By means of microsurgical lymph cannulation human lymph derived from normal skin was sampled. Cells were isolated and processed for immunocytochemistry, electron microscopy, flow cytometry and functional assays. The majority of the cells, (62%), were T cells. The other cells comprised CD1a⁺ cells (7%), monocytes/macrophages (8%), and B cells (1%); the remainder were erythrocytes or uncharacterized cells. The CD1a⁺ cells reacted with antibodies against protein S-100, HLA-DR, the Lag antigen, CD4, CD11a, CD11b, CD18, CD25, CD40, CD54, CD80 and CD86. Interestingly, a small prolow portion the of CD1a⁺ cells (about 5%) reacted with an antibody to CD14. The CD1a⁺ cells did not react with an antibody against human follicular dendritic cells nor were they CD19-, CD23-, E-cadherin- or factor XIIIa-positive. Both allogenic and antigen-specific T cell proliferation stimulated by antigen-presenting lymph cells were strongly inhibited by adding anti-CD80 and anti-CD86 antibodies. By electron microscopy Birbeck granules were detected in only 22% of the CD1a⁺ lymph cells and these cells exhibited an extensive ruffling of the surface. These findings demonstrate that CD1a⁺ lymph cells, which do not express the dermal dendritic cell marker factor XIIIa, resemble dendritic cells formerly designated as ‘veiled’ as well as lymphoid dendritic cells, suggesting that after migration to the regional lymphoid organs, Langerhans cells form a more differentiated population of dendritic cells specialized in sensitizing T lymphocytes. Our results add further support to the view that resident Langerhans cells may be precursors of lymphoid dendritic cells acquiring the final phenotype in the microenvironment of the lymph node. Received: 30 July 1998 / Received after revision: 30 September 1998 / Accepted: 19 October 1998     

Cytokine expression in psoriatic skin lesions during PUVA therapy

To determine whether an improvement in skin lesions as a result of PUVA therapy may be correlated with changes in cytokine patterns, RT-PCR amplification was used to compare the levels of IL-2, IL-6, IL-8, IL-10, TNF-α and IFN-γ cytokine mRNA expression in serial biopsies from three chronic plaque psoriatic patients. In each case, 3-mm punch biopsies were taken from lesional skin before and during 2–28 days of treatment with PUVA. Total mRNA was extracted from each biopsy, cDNA synthesized, and then amplified by 35 cycles of PCR using cytokine-specific primers. The specificity of the PCR products was confirmed by the Southern blot technique. Substantial levels of specific mRNA for each of the cytokines studied was present in the lesions prior to treatment. In two of the three patients who responded well to PUVA, a reduction in all the cytokines including IL-10 was observed compared with baseline levels. In contrast, PUVA proved to be ineffective in clearing the psoriasis of the third patient whose skin lesions worsened during the course of treatment. This was accompanied by an increase in IFN-γ but not of the other cytokines investigated, above the pretreatment level. This study showed an association between PUVA-induced resolution and decreases in the levels of various cytokines highly expressed in psoriatic lesions. Received:14 August 1995     

CD103+T细胞在银屑病皮损中的表达

目的：明确银屑病患者皮肤CD103+T细胞的表达及其与银屑病严重程度的关系。方法：免疫组化检测29例银屑病患者皮损和非皮损皮肤及6名健康对照皮肤中表皮及真皮CD103+T细胞的表达。计算银屑病患者PASI值。结果：CD103+T细胞主要在真皮表达。银屑病患者皮损和非皮损真皮中每个高倍视野CD103+T细胞百分率分别为（26.06%±11.72）%和（12.82±4.5）%（P<0.05）;健康人对照皮肤真皮内CD103+T细胞百分率为（7.47±1.3）%,明显低于银屑病非皮损区（P<0.05）。银屑病患者皮损中,CD103+T的表达与PASI值正相关（P<0.05）。 结论：真皮中CD103+T细胞可能与银屑病的发病及严重程度有关。     

Nucleolar ultrastructure in psoriatic skin lesions

Keratinocytes in psoriatic skin lesions were studied to provide more information on the nucleolar ultrastructure in these cells. In contrast to normal keratinocytes, nucleoli in the keratinocytes of the uppermost layer of stratum granulosum (intermedium) of psoriatic lesions were characterized by the absence of the segregation of nucleolar components. In addition, the keratinocytes without keratohyalin of the uppermost layer of stratum intermedium of psoriatic lesions did not contain degranulated nucleoli. Such observations indicate an alteration of the maturation processes in the keratinocytes of the psoriatic skin lesions and the characteristic nucleolar changes apparently represent the morphological expression of the altered inhibition of the nucleolar (preribosomal) RNA synthesis.     

In situ localization of CD83-positive dendritic cells in psoriatic lesions

BACKGROUND: Dendritic cells (DC) are considered to be the most potent antigen-presenting cells, and CD83 is expressed at a high level on immune-competent, activated and mature DC. Although changes in the number or localization of mature and activated CD83+ DC could be expected in psoriasis, there is little information on such changes. AIM: Morphological identification of CD83+ DC in psoriatic skin lesions. MATERIALS AND METHODS: Immunohistochemical staining was performed in 5 specimens of psoriasis vulgaris and 6 specimens of pustular psoriasis. Formalin-fixed, paraffin-embedded sections were used for examination in this study. The skin sections were pretreated with 0.1% trypsin for 60 min at 37 degrees C prior to immunostaining for CD83. RESULTS: A small but significant subpopulation of CD83+ DC was found in the upper dermis. In addition, CD83+ DC were occasionally scattered in the epidermis. The most common distribution pattern of CD83+ DC was as clusters with mononuclear lymphoid cells in the upper dermis. CD83+ DC were in close contact with lymphocytes. High-intensity staining of CD83 antigens was detected not only on the surface, but also in the cytoplasm of DC. CONCLUSION: These results indicate that activated and mature CD83+ DC may play a role in the immune response in psoriasis and provide in vivo support for the concept that CD83+ DC provide signals for direct intralesional T cell activation.     

The presence of tryptase-positive and bikunin-negative mast cells in psoriatic skin lesions

Human mast cells are well known to produce a serine protease, tryptase, which appears to play a pathogenic role in various skin inflammations. It was previously reported that a rat homologue of bikunin may inhibit tryptase activity. Various type of cells (i.e. keratinocytes) are able to produce this protein inhibitor, it still remains unclear if bikunin is present in dermal inflammatory milieu, in which mast cells, through secretion of tryptase, play an inflammatory role. Therefore, the purpose of the present study was to exploit expression and production of bikunin in dermis and dermal constituents. We first compared the dermal mast cells in psoriatic lesions with those in lesional skin of atopic dermatitis or of chronic eczema by use of immunoelectron microscopy and immunohistochemical analyses using antibodies to bikunin and tryptase. Then, we tested what kinds of cytokines may regulate the de novo synthesis of bikunin. To do so, RNA was extracted from a human mastocytic cell line, HMC-1, reverse-transcribed, and semiquantitative RT-PCR was performed using primers specific for bikunin. With immunoelectron microscopy, bikunin was found to localize on the cell membrane, while tryptase was in the secretary granules of the mast cells. In psoriatic lesions, around 70% of dermal mast cells were positive for both tryptase and bikunin, and the remaining was mostly positive for tryptase, but the expression of bikunin was under the detection limit of the experimental setting. This observation was seen in only psoriatic lesions, even in almost cured lesions, while in atopic dermatitis or chronic eczema only mast cells doubly positive for bikuin and tryptase were seen. In HMC-1, bikunin was constitutively expressed at an mRNA level, which was upregulated by stimulation with interleukine-4, but was suppressed by interferon-γ. Bearing in mind the concept that in psoriasis local cytokine milieu is shifted toward a Th1 pattern (predominant secretion of interferon-γ), tryptase-positive, bikunin-negative mast cells may be induced.     

银屑病患者皮损间充质干细胞环状RNA表达异常研究

目的 探讨环状RNA（circRNA）在银屑病发病中的作用。方法 分离、培养15例银屑病患者皮损及15例健康对照皮肤间充质干细胞（MSC）,用流式细胞仪及多向分化法进行鉴定。用RNA测序法检测circRNA表达,并进行详细的生物信息学分析。挑选7个差异表达的circRNA构建circRNA-microRNA相互作用网络,从中挑选3个与其相关的microRNA进行qRT-PCR验证。银屑病患者组和对照组qRT-PCR验证结果采用两独立样本t检验。结果 RNA测序结果显示,共检测到6 323个circRNA,其中3 227个为本实验首次发现。与对照组相比,患者组中有129个circRNA呈差异表达,其中123个表达上调,6个表达下调。挑选7个差异circRNA进行circRNA-microRNA相互作用预测,显示与这7个circRNA关系密切的银屑病相关microRNA,包括miR-17-5p、miR-30e-5p、miR-142-3p/5p、miR-369-3p、miR-184、miR-4490、miR-654-3p、miR-423-5p等。qRT-PCR也证实,这7个差异circRNA在患者组也表达上调。与对照组相比,挑选的3个microRNA在银屑病皮损中低表达（t值分别为3.993、3.217、2.918,均P < 0.05）。结论 银屑病皮损MSC circRNA表达异常,并可能参与了银屑病发病。     

Mesenchymal stem cells in psoriatic lesions affect the skin microenvironment through circular RNA

Psoriasis is an autoimmune skin disease. Our previous studies revealed abnormal immune regulation of skin mesenchymal stem cells (S‐MSCs) in psoriatic lesions. Circular RNA (circRNA) molecules were recently discovered as a new class of non‐coding regulatory RNAs. Their role in the pathogenesis of psoriasis has not yet been studied. To explore potential circRNA‐mediated mechanisms of S‐MSCs in the pathogenesis of psoriasis, we sequenced mRNAs and circRNAs of MSCs from normal skin and psoriatic lesions, followed by functional prediction and interaction analyses. In total, 129 circRNAs were differentially expressed, including 123 up‐regulated and 6 down‐regulated circRNAs, in MSCs from psoriatic lesions. Pathway analysis showed that the genes significantly down‐regulated in psoriatic as compared to normal S‐MSCs were mainly involved in JAK‐STAT signalling. According to a circRNA‐miRNA‐mRNA interaction network, the expression of circRNAs associated with these mRNAs was also down‐regulated in MSCs of psoriatic skin lesions. Knockdown of the circRNA gene chr2:206992521|206994966 reduced the capacity of S‐MSCs to inhibit T‐cell proliferation upon co‐culture in normal as well as lesion‐derived S‐MSCs. Secreted‐cytokine profiles (IL‐6, IL‐11 and hepatocyte growth factor) were also similar in normal and lesion‐derived S‐MSCs after circRNA knockdown. Thus, the circRNA chr2:206992521|206994966 in S‐MSCs from psoriatic lesions affects the activity of T lymphocytes in local lesions by influencing their cytokine secretion. Taken together, our findings indicate that circRNA mediates the role of S‐MSCs in the pathogenesis of psoriasis.     

Fc gamma-receptors and HLA-DR antigens on endothelial cells in psoriatic skin lesions

Receptors for the Fc-part of IgG (FcR) and HLA-DR antigens on endothelial cells in normal and lesional skin from patients with psoriasis were studied in cryostat sections, using soluble immune complexes and monoclonal antibodies. FcR and HLA-DR antigens were detected on endothelial cells of dermal vessels both in sections of normal and lesional skin. The expression of FcR varied from one vessel to another and on endothelial cells within one and the same vessel. The expression of FcR and HLA-DR antigens was enhanced in sections of lesional skin compared with normal skin and most pronounced in lesional skin from active psoriasis. The enhanced expression may be mediated by interferon produced in psoriatic lesions. The presence of FcR and HLA-DR antigens on endothelial cells adds further evidence of he involvement of these cells in immune processes in the skin.     

CD1a-positive dendritic cells transport the antigen DNCB intracellularly from the skin to the regional lymph nodes in the induction phase of allergic contact dermatitis

Abstract Dendritic cells are potent stimulators of T cell-mediated immune responses. In contact hypersensitivity reactions in animals dendritic cells have been reported to transport antigens to the regional lymph nodes. In this study we investigated whether skin-derived dendritic cells transport contact antigens via the afferent lymph in humans. By means of a microsurgical technique lymph cells were collected after painting a defined skin region with a 2% concentration of the sensitizing agent 2,4-dinitrochlorobenzene on the leg of 14 volunteers. There was no significant change in flow, output or composition of cells after antigen painting. Using flow cytometric analysis we were able to detect the antigen in CD1a⁺ dendritic cells of the afferent lymph 15–25 h after antigen application. The antigen could only be detected after permeabilizing the dendritic cells, indicating that the main part of the antigen is transported intracellularly and not on the surface of these cells. Further analysis of cell surface antigens such as CD80, CD86, HLA-DR, CD11a, CD14, CD23, CD25 and CD54 revealed that in the course of cutaneous sensitization the phenotype of the dendritic cells was not altered in the afferent lymph. These results provide direct evidence that during the induction phase of allergic contact dermatitis in humans antigen-bearing dendritic cells internalize the antigen and migrate from the skin via the afferent lymph vessels to the lymph nodes. Received: 18 January 2001 / Revised: 3 May 2001 / Accepted: 11 July 2001     

Heterogeneous distribution of lipoxygenase products in psoriatic skin lesions

Summary Several biologically active lipoxygenase products or arachidonic acid (AA) have been demonstrated in psoriatic skin lesions. The purpose of the present study was to determine the amounts of the different lipoxygenase products simultaneously in psoriatic skin. Slices of psoriatic skin were obtained at different levels with a keratome. Extracted lipids were identified by high performance liquid chromatography, UV-absorption spectrum, radioimmunoassay, and chemokinesis. Leukotriene B₄ (LTB₄), and 12- and 15-hydroxy-eicosatetraenoic acid (HETE) were detected in most psoriatic lesions. However, there was a remarkable variation from lesion to lesion. The biopsy specimens contained: 276.2±126.0 pg/g wet tissue of LTB₄, 3,130.0±2,898.0 ng/g wet tissue of 12-HETE, and 3,633.0±1,692.0 ng/g wet tissue of 15-HETE. No correlation was found between the levels of the different lipoxygenase products. The content of each of the identified lipoxygenase products was higher in the superficial part of the biopsy specimen consisting of approximately two-thirds of the epidermis plus papillary dermis than in the lower part consisting of approximately one-third of the epidermis plus some reticular dermis. Also, there was a great variation from one anatomical region to another within the same patient. Because these lipoxygenase products possess different biological activities, the variation in their occurrence may be important for understanding their potential role in psoriasis. To determine which lipoxygenase products may be of pathogenic importance, analysis of early psoriatic lesions is warranted.     

Blood flow in psoriatic skin lesions: the effect of treatment

Laser Doppler velocimetry (LDV) was used to assess the effect of treatment upon cutaneous blood flow in psoriatic skin lesions. The resolution of two separate plaques in each of seven subjects was followed. Six of the subjects received the Goeckerman regimen, the seventh was treated with psoralen-ultraviolet A (PUVA) therapy. Control LDV readings were taken from uninvolved skin sites during the treatment period. Cutaneous blood flow in the psoriatic lesions of the Goeckerman-treated patients decreased to levels comparable to those in uninvolved skin early in the course of treatment and significantly preceeded the observed clinical resolution from 4 to 8 days after initiation of therapy (P less than 0.05). Visible flare-ups sometimes appeared when patients were untreated (over a weekend, for example) and these eruptions were accompanied by a transient elevation of LDV readings. Perfusion of the lesions of the PUVA-treated patient remained consistently higher than perfusion of uninvolved skin despite clinical healing. In a separate series of experiments, blood flow at the extensor surface of the forearm was measured in the skin of normal subjects, the uninvolved skin of psoriatic patients and the untreated lesional skin of psoriatic patients. While the lesional skin had significantly higher perfusion levels than uninvolved psoriatic or normal control skin (P less than 0.01), there was no significant difference between blood flow to the uninvolved psoriatic skin of psoriatics and that to the skin of healthy controls.     

Subpopulations of mononuclear cells in microscopic lesions of psoriatic patients. Selective accumulation of suppressor/cytotoxic T cells in epidermis during the evolution of the lesion

The age of microscopic lesions in psoriatic subjects was assessed from the stacking characteristics in the horny layer and related to type and density (cells/tissue volume) of mononuclear cells in the epidermis and the dermis determined by immunoperoxidase methods using monoclonal antibodies. Pan T cells (Lyt-2+, Lyt-3+, Leu-4+, OKT3+), T helper cells (Leu-3a+, OKT4+), T suppressor/cytotoxic cells (Leu-2a+, OKT8+), Ia+ cells and monocytes (OKM2+, BRL alpha mono+) were determined in epidermis and dermis. The psoriatic lesion was divided into regions underneath a parakeratotic and an orthohyperkeratotic/hypergranular portion of the horny layer and contrasted with perilesional and uninvolved psoriatic skin as well as with healthy skin. In the various regions and skin layers, the cell density was highest in parakeratosis and decreased toward normality with decreasing histologic abnormality. The relation between epidermal and dermal cell densities of the T-cell subsets was modified in the involved psoriatic skin with a selective preponderance of T suppressor/cytotoxic cells in the epidermis. The accumulation was present in the youngest lesion found (3 days) and cell densities were unchanged in older lesions. The findings suggests that the altered relationship in the subsets of T cells has an important role during the induction and progress of the psoriatic process in the skin.     

Monolayer culture of cells from psoriatic lesions

A method for culturing monolayers of cells from psoriatic lesions is described. Cultures have been maintained for 3 to 8 weeks. Apart from keratinocytes, melanocytes and non-pigmented dendritic cells, two types of cell were observed which are not commonly seen in normal skin, viz. a cell with a basket weave type of cytoplasm and a cell with a very long, up to 500 μm, cytoplasmic extension.     

Rise in dermal CD11c+ dendritic cells associates with early-stage development of psoriatic lesions

There is limited information available regarding the phenotype and function of leukocytes involved in the earliest stages of psoriatic lesion development. In this study, we examined the presence of different types of leukocytes in psoriatic point lesions collected at three 1-week interval time points from a recent and simultaneously formed group of point lesions. The cells were quantified and compared with K16 expression and epidermal thickness, both typically increased in this disease and considered as hallmarks. We found a significant correlation between K16(+) cell increment and the increase in epidermal thickness in the timeframe of 14 days. The change in CD3(+), CD4(+), and CD8(+) T-cell numbers in the dermis showed a significant association with these two features from d7 to d14, whereas in the epidermis only CD8(+) T cells demonstrated a significant correlation. Remarkably, the relationship between T cells and disease progression was preceded by a significant correlation of CD11c(+) dendritic cells (DCs) with K16 expression and epidermal thickness from baseline onwards. Interestingly, there was also a numeric correlation of CD11c(+) DCs with the CD3(+) T-cell shifts from d7 to d14. A significant correlation was also found between dermal CD14(+) cells and K16 expression from d7 to d14. BDCA-2(+) plasmacytoid DCs were absent in non-lesional skin, but found at low numbers in most lesions. The change in plasmacytoid DC or neutrophil numbers did not correlate with lesion development. In conclusion, our study suggests a relevant role for T cells, and in particular dermal CD11c(+) DCs, in the earliest stage of psoriatic lesion development.     

Antigen-independent expansion of T cells from psoriatic skin lesions: phenotypic characterization and antigen reactivity

The pathogenesis of psoriasis appears to depend on T cells, which have been proposed to mediate the disease through an autoimmune process. To test this hypothesis we have propagated four T-cell lines from biopsies of psoriatic skin lesions by antigen-independent methods. Flow cytometric immunophenotyping showed the lines to be composed mainly of CD4-positive, αβ-cell receptor (TCR)-positive cells, which secreted a cytokine profile suggestive of predominant T-helper type 1 (Th1) status. Analysis of TCR variable region (Vβ) usage revealed two- to eight-fold increases in the expression of certain Vβ species in lesional lines as compared with autologous peripheral blood mononuclear cells (PBMC), with the increased Vβ species being expressed on more than 5% of cells in two of the lines. Lines were also used to test for responses to a range of epidermal antigen preparations in the presence of irradiated autologous PBMC as antigen-presenting cells. The lines failed to proliferate in response to psoriatic lesional stratum corneum extracts, dispase-separated normal human epidermal extracts, and an epidermal keratin preparation before and after trypsinization, in spite of good proliferative responses to anti-CD3 which indicated that the lines were not anergic. In addition, the lines and PBMC from normal volunteers and the patients with psoriasis gave little or no response to recombinant streptococcal M protein. Thus, in spite of accumulating evidence for selective expansion of certain Vβ-expressing T cells in psoriatic lesions, epidermal autoantigens have not been identified by using a bioassay which depended largely on the proliferation of lesional CD4-positive cells. The role of streptococcal M protein, which bears some homology with epidermal keratin is also open to question, at least in chronic plaque psoriasis. Further work is therefore required to obtain direct evidence that autoimmune processes are important in the pathogenesis of chronic plaque psoriasis.