Langerhans cell hyperplasia and IgE expression in canine atopic dermatitis

Langerhans cells appear to be critical for IgE-mediated allergen capture and presentation in human atopic dermatitis. The present study sought to determine whether epidermal (i.e Langerhans cells) and dermal dendritic cells in the skin of dogs with atopic dermatitis are hyperplastic and expressed surface IgE. Frozen sections of lesional or nonlesional atopic and normal control canine skin were immunostained with CD1a-, CD1c-, and IgE-specific monoclonal antibodies. The enumeration of cells was performed by morphometry in both the epidermis and the dermis. Cell counts were compared with each individual’s total serum IgE levels. Higher numbers of epidermal and dermal dendritic cells were present in atopic dogs than in normal control animals. Epidermal Langerhans cell counts were significantly higher in lesional than in nonlesional atopic specimens. IgE⁺ dendritic cells were observed in lesional atopic epidermis and dermis, and nonlesional atopic dermis, but not in normal control skin specimens. The percentages of IgE⁺ dendritic cells were correlated with each patient’s total serum IgE levels. These results demonstrate dendritic cell hyperplasia and IgE expression in canine atopic dermatitis. Increased epidermal Langerhans cell counts in lesional specimens suggest an epidermal allergen contact in canine atopic dermatitis.     

Antigen-presenting cells in essential fatty acid-deficient murine epidermis: keratinocytes bearing class II (Ia) antigens may potentiate the accessory cell function of Langerhans cells.

Essential fatty acid deficiency (EFAD) is a useful model for studying the role of (n-6) fatty acid metabolism in normal physiology. Because cutaneous manifestations are among the earliest signs of EFAD and because abnormalities in the distribution and function of tissue macrophages have been documented in EFAD rodents, we studied the distribution and function of Class II MHC (Ia) antigen-bearing cells in EFAD C57B1/6 mouse epidermis. Immunofluorescence studies revealed 1.9-9.6 (mean +/- SEM = 5.2 +/- 2.6) times more class II MHC (Ia) antigen-bearing epidermal cells in suspensions prepared from EFAD as compared to normal skin. Analysis of epidermal sheets demonstrated similar numbers of dendritic Ia+ and NLDC145+ cells in EFAD and normal epidermis, however. This discrepancy occurred because some keratinocytes in EFAD epidermal sheets expressed class II MHC (Ia) antigens, whereas keratinocytes in normal mouse epidermis did not. Two-color flow cytometry confirmed that all Ia+ cells in normal epidermis are Langerhans (Ia+ NLDC145+) cells, whereas Ia+ cells in EFAD epidermis are comprised of Langerhans cells and a subpopulation of keratinocytes (Ia+ NLDC145-). Similar levels of Ia antigens were expressed on EFAD and normal Langerhans cells. EFAD and normal epidermal cells were also compared in several in vitro assays of accessory cell function. Epidermal cells prepared from EFAD C57B1/6 mice present the protein antigen DNP-Ova to primed helper T cells more effectively than epidermal cells prepared from normal animals. EFAD epidermal cells are also more potent stimulators of T cells in primary and secondary allogeneic mixed lymphocyte-epidermal cell reactions than normal epidermal cells. The functional differences between EFAD and normal epidermal cells do not appear to result from increased cytokine release or decreased prostaglandin production by EFAD epidermal cells. In view of these findings and the observation that the antigen-presenting cell activity of EFAD epidermal cells correlates with the number of Ia+ keratinocytes in epidermal cell preparations, Ia+ keratinocytes (in the presence of Langerhans cells) may potentiate cutaneous immune responses in vitro and perhaps in vivo as well. These results also suggest that (n-6) fatty acids or metabolites of (n-6) fatty acids are involved in regulating the expression of class II MHC (Ia) antigens by keratinocytes in vivo.     

Calcipotriol inhibits the proliferation of hyperproliferative CD29 positive keratinocytes in psoriatic epidermis in the absence of an effect on the function and number of antigen-presenting cells

The aim of this study was to elucidate some of the possible mechanisms of action of the vitamin D analogue calcipotriol in vivo . Calcipotriol is finding increasing use in the treatment of psoriasis, but the primary target cell in vivo has not yet been identified. We treated psoriatic patients and healthy volunteers with calcipotriol and placebo ointment for 4 and 7 days, and obtained epidermal cell suspensions from treated areas. Epidermal cells were cocultured with autologous T cells, isolated from peripheral blood, in the absence or the presence of a classical antigen or a superantigen. In both psoriatic and normal skin, calcipotriol treatment did not alter the capacity of epidermal antigen-presenting cells to stimulate the proliferation of autologous T cells, either in the absence or in the presence of exogenous antigen. Epidermal cell suspensions were analysed further by staining for infiltrating leucocytes (CD45+) and Langerhans cells (CD1a+). Flow cytometric analyses showed that calcipotriol did not alter the number of CD45+ cells or Langerhans cells in psoriatic skin. These results indicate that calcipotriol does not alter either the number or the function of epidermal antigen-presenting cells in psoriatic epidermis. In contrast, we found that calcipotriol significantly inhibited the proliferation of epidermal cells isolated from psoriatic skin after in vivo treatment, as determined by propidium iodide staining and flow cytometry. More specifically, we stained for CD29+ keratinocytes and found an even more significant reduction in proliferative capacity. This cell type contains the population of hyperproliferative keratinocytes in psoriatic epidermis. In conclusion, calcipotriol seems to act via an inhibitory effect on hyperproliferative basal keratinocytes of psoriatic epidermis, rather than via an effect on infiltrating leucocytes, including antigen-presenting cells.     

Somatostatin regulates tight junction function and composition in human keratinocytes

Please cite this paper as: Somatostatin regulates tight junction function and composition in human keratinocytes. Experimental Dermatology 2010; 19 : 888–894. Abstract: Somatostatin (SST) is a regulatory peptide hormone that acts through five different G protein‐coupled receptors (SSTR1‐5). Whereas expression of all five SSTR subtypes in epidermis has been shown, the biological relevance of the SST/SSTR system in the skin is completely unknown. We show here that SST is expressed in human skin and is present in a subset of Merkel cells and dendritic cells as well as in keratinocytes. We focused further on the somatostatin receptor subtype 3 (SSTR3) and its interacting protein MUPP1, as both were found to be localized at cellular junctions in epidermal keratinocytes. MUPP1 is a component of tight junctions (TJs); these cell–cell junctions contribute to barrier function of the paracellular pathway in cultured keratinocytes. We provide evidence that SSTR3 and MUPP1 interact in primary cultured human keratinocytes at high Ca²⁺ conditions. Interestingly, SST, presumably via SSTR3/MUPP1, regulates TJ permeability in cultured keratinocytes. During long‐term treatment of human keratinocytes, SST also affects the expression of distinct TJ proteins such as claudin‐4. Our data are the first example of a peptide hormone regulating TJ functionality and composition in human keratinocytes, suggesting that control via peptide hormones provides the possibility to regulate the TJ barrier characteristics of the skin.     

Glucocorticoid receptor localization in human epidermal cells

Glucocorticoids, which are widely used in therapy, exert their immunosuppressive actions through specific receptors. These receptors have been characterized in cultured human skin fibroblasts and keratinocytes, but their localization in vitro and in vivo has not been established. To determine the tissue and cellular distribution of glucocorticoid receptors (GR), two specific polyclonal rabbit anti-human GR antibodies were used to detect these receptors in skin biopsy specimens, in freshly isolated and cultured human epidermal cells and in keratinocyte cell lines. Immunoreactive GR were only faintly detected in normal and abnormal differentiated cells and as well as those in the stratum granulosum and corneocytes. These immunolocalization studies were confirmed by fluorescence cell sorter analysis of isolated basal and suprabasal keratinocytes. Immunoreactive GR were highly expressed in normal cultured human keratinocytes, Langerhans cells and several cell lines whereas they were less expressed in melanocytes. Based upon these results the main targets of glucocorticoids in the epidermis appear to be basal and Langerhans cells. Received: 6 February 1995     

吲哚胺2,3-双加氧酶在尖锐湿疣皮损中表达的研究

目的 分析尖锐湿疣组织中吲哚胺2,3-双加氧酶(IDO)水平,研究其局部代谢色氨酸的能力.方法 免疫组化法观察尖锐湿疣患者皮肤IDO蛋白表达情况,计数IDO阳性细胞的比例.免疫荧光观察IDO(+)细胞与树突细胞的关系.分离对照皮肤角质形成细胞和疣体上皮细胞,色氨酸体外孵育后检测上清液中色氨酸代谢产物犬尿氨酸的浓度以反映细胞代谢色氨酸的能力.结果IDO(+)细胞在正常皮肤中非常少,但在疣体表皮中却大量聚集.疣体内IDO(+)细胞/总体细胞48.3%±15.4%显著高于正常皮肤5.2%±2.4%,差异有统计学意义(P<0.05).IDO(+)细胞的荧光信号和皮肤朗格汉斯细胞并不重合,提示来源于疣体表皮细胞.从疣体组织分离的上皮细胞在体外代谢色氨酸的能力强于健康对照皮肤中分离的表皮细胞.结论 尖锐湿疣疣体中存在大量的IDO(+)细胞,这些细胞可能参与尖锐湿疣的发病.     

Absence of expression of class II major histocompatibility complex determinants on keratinocytes in bullous pemphigoid

Aberrant expression of class II products of the major histocompatibility complex (HLA-D locus antigens) occurs on keratinocytes in several inflammatory dermatoses and on thyroid epithelial cells in autoimmune thyroiditis. The functional significance of aberrant HLA-D expression is unclear but it has been hypothesized that epithelial cells bearing these determinants may act as antigen-presenting cells for autoantigens. The aim of the present study was to investigate the pathogenesis of bullous pemphigoid using immunohistochemical methods to determine whether the HLA-D locus antigens are aberrantly expressed on keratinocytes in lesional and uninvolved skin. A panel of monoclonal antibodies to each of the HLA-D subregions (DR, DP and DQ) and to Langerhans cells was used. Epidermal expression of the HLA-D locus antigens was similar in patients and controls, and there was no significant increase in expression in lesional skin compared with uninvolved skin in six out of nine patients. In three out of nine patients slight enhancement of epidermal HLA-D expression in lesional epidermis corresponded to increased Langerhans cells rather than expression on keratinocytes. HLA-D locus antigens are absent from keratinocytes in bullous pemphigoid skin and aberrant expression of these determinants cannot therefore be implicated in antigen presentation.     

Local effects of TL01 phototherapy in psoriasis

The mechanisms whereby narrowband ultraviolet B (UVB) (311-313 nm, TL01) phototherapy are effective in psoriasis may differ from those occurring in broadband UVB phototherapy. In the present study, changes in epidermal cells as a result of TL01 therapy were assessed in the skin of patients with psoriasis. The non-lesional skin of five subjects with plaque psoriasis was biopsied before and after a series of 12 whole-body TL01 treatments. Following appropriate staining of skin sections, the numbers of p53-positive keratinocytes, sunburn cells and Langerhans cells in the epidermis were counted. TL01 therapy induced a threefold increase in the number of p53-positive epidermal cells, a 12-fold increase in sunburn cells and a twofold decrease in Langerhans cells. The increase in epidermal p53 expression and apoptosis of keratinocytes together with the depletion of Langerhans cells in the non-lesional skin of psoriasis patients are likely to contribute to the effectiveness of TL01 phototherapy.     

Expression of OKM5 antigen on epidermal cells in mycosis fungoides plaque stage

To characterize and quantitate potential antigen-presenting cell subsets in the epidermis of patients with cutaneous T-cell lymphoma, epidermal cells in suspension were obtained from involved and uninvolved skin. Involved epidermis contained increased numbers of OKT6+HLA-DR+ Langerhans cells and a variable number of OKM5+ epidermal cells (ECs) in all mycosis fungoides (MF) patients tested (N = 14). The OKM5+ EC population from involved epidermis of MF patients were heterogeneous and comprised both OKM5+HLe1- keratinocytes and OKM5+HLe1+ leukocytes. Uninvolved epidermis, in 6 of 14 patients with MF, contained a small number of OKM5+ leukocytes; however, no OKM5+ keratinocytes were detected. Neither OKM5+ leukocytes nor OKM5+ keratinocytes were detected in the epidermis obtained from healthy controls. The increased number of potential antigen-presenting cells, that is, OKT6+HLA-DR+ Langerhans cells and OKM5+HLA-DR+ monocytic leukocytes, in the epidermis of patients with MF may be important for the activation of abnormal T cells contained within the epidermis of these patients. Such activated T cells may release gamma-interferon and induce expression of both HLA-DR and OKM5 antigens on keratinocytes. OKM5+ keratinocytes are present in the epidermis of patients with MF, but not in normal skin, and may thus play a role in the pathogenetic mechanisms of mycosis fungoides by recruitment of immunocompetent cells to the epidermis.     

结合珠蛋白在正常人表皮细胞及HaCaT细胞中的表达

目的 检测正常人表皮细胞及HaCaT细胞中结合珠蛋白(Hp)mRNA及蛋白的表达。方法 用原位杂交及RT-PCR法检测正常人表皮细胞及HacaT细胞中Hp mRNA的表达，用免疫组化法检测正常人表皮中Hp的表达；用免疫组化法及蛋白质免疫印迹法检测HaCaT细胞中Hp的表达。结果 在正常人表皮角质形成细胞(KC)及HaCaT细胞中Hp mRNA表达阳性；正常人表皮朗格汉斯细胞(LC)中Hp mRNA表达阴性；正常人表皮内可见Hp阳性的树突状细胞。用免疫组化法在每个HaCaT细胞胞质中未见明显Hp染色，但用蛋白质免疫印迹法在HacaT细胞中检测到Hp蛋白。结论 正常人KC及HaCaT细胞有合成Hp能力，正常人表皮LC无合成Hp的能力，在HaCaT细胞中有少量Hp表达。     

Expression of activated MEK1 in differentiating epidermal cells is sufficient to generate hyperproliferative and inflammatory skin lesions

Epidermal activation of Erk MAPK is observed in human psoriatic lesions and in a mouse model of psoriasis in which beta1 integrins are expressed in the suprabasal epidermal layers. Constitutive activation of the upstream kinase MEK1 causes hyperproliferation and perturbed differentiation of human keratinocytes in culture. It is not known, however, whether Erk activation in differentiating keratinocytes is sufficient to trigger hyperproliferation of basal keratinocytes and a skin inflammatory infiltrate. To investigate this, we expressed constitutively active MEK1 in the suprabasal epidermal layers of transgenic mice. Proliferation in the epidermal basal layer was stimulated and epidermal terminal differentiation was perturbed. Some older mice also developed papillomas. There was a large increase in T lymphocytes, dendritic cells, and neutrophils in the skin. The effects of suprabasal MEK1 on basal keratinocytes and leukocytes, cells that were transgene negative, suggested that MEK1 activity might stimulate cytokine release. Transgenic keratinocytes expressed elevated IL-1alpha and crossing the mice with mice overexpressing the IL-1 receptor in the epidermal basal layer led to exacerbated hyperproliferation and inflammation. These data suggest that activation of MEK1 downstream of beta1 integrins plays an important role in epidermal hyperproliferation and skin inflammation.     

Normal keratinocytes express Toll-like receptors (TLRs) 1, 2 and 5: modulation of TLR expression in chronic plaque psoriasis

BACKGROUND: Toll-like receptors (TLRs) are part of the innate immune system involved in the response to microbial pathogens. TLR2 recognizes various ligands expressed by Gram-positive bacteria, while TLR3, TLR4 and TLR5 are specific for double-stranded RNA, Gram-negative lipopolysaccharides and bacterial flagellin, respectively. OBJECTIVES: To determine, firstly, whether epidermal keratinocytes of normal skin express TLRs and, secondly, whether modulation of TLR expression occurs in psoriasis, an inflammatory skin disease associated with certain microorganisms such as streptococci, staphylococci and yeasts. METHODS: Eight samples of normal, and 15 samples of lesional and nonlesional psoriatic skin were stained with polyclonal antibodies specific for TLR1-5 using an avidin-biotin-peroxidase technique. RESULTS: Epidermal keratinocytes in normal skin constitutively expressed TLR1, TLR2 and TLR5, while TLR3 and TLR4 were, in most cases, barely detectable. Cytoplasmic TLR1 and TLR2 were expressed throughout the epidermis, with higher staining of the latter on basal keratinocytes, while TLR5 expression was concentrated in the basal layer. In contrast, in lesional epidermis from patients with psoriasis, TLR2 was more highly expressed on the keratinocytes of the upper epidermis than on the basal layer, while TLR5 was downregulated in basal keratinocytes compared with corresponding nonlesional psoriatic epidermis. In addition, nuclear TLR1 staining was observed in the upper layers of both nonlesional and lesional psoriatic epidermis, but not in that of normal skin. CONCLUSIONS: These findings suggest that TLRs expressed by epidermal keratinocytes constitute part of the innate immune system of the skin. The relevance of altered keratinocyte TLR expression in psoriasis remains to be determined.     

Self‐renewal capacity of human epidermal Langerhans cells: observations made on a composite tissue allograft

Abstract: Epidermal Langerhans cells (LC) are dendritic, antigen‐presenting cells residing within mammalian epidermis and mucosal epithelia. When massively depleted, they are replaced by cells of bone‐marrow origin. However, their renewal within normal skin under steady‐state conditions is not precisely known. We observed that epidermal LC within a human hand allograft remain stable in the long term (10 years) and are not replaced by cells of recipient’s origin; furthermore, we observed a Langerhans cell in mitosis within the epidermis 8 years postgraft. These results show that under almost physiological conditions, human LC renew in the epidermis by local mitoses of preexisting cells.     

Epidermal cytokines and their roles in cutaneous wound healing

Cytokines are small proteins or glycoproteins which are synthesized and secreted by a variety of cell types. Through binding to specific receptors on target cells, these hormone-like products regulate many normal cell activities, including growth and differentiation, migration and immune functions. Within the epidermis, keratinocytes are the major source of cytokines along with melanocytes and Langerhans cells. In response to a variety of injurious stimuli, including ultraviolet irradiation and cutaneous wounding, epidermal keratinocytes may release a number of these regulatory molecules which can then interact directly with receptors on inflammatory cells. Epidermal cytokines can therefore play an important role in the wound-healing process by recruiting polymorphs and monocytes and in encouraging deposition of extracellular matrix proteins by fibroblasts. Keratinocytes can themselves respond to keratinocyte-derived cytokines by dividing and migrating over the wound surface before differentiating into a new stratified epidermis. This review presents the evidence of the production of cytokines by human keratinocytes and their role in the healing of skin wounds.     

Occurrence of Langerhans cells and expression of class II antigens on keratinocytes in malignant and benign epithelial tumors of the skin: an immunohistopathologic study with monoclonal antibodies

We used an avidin-biotin complex immunoperoxidase technique with various monoclonal antibodies to determine Langerhans cell densities, class II antigen expression on keratinocytes, and phenotypes of other infiltrating cells in several malignant and benign epithelial tumors of the skin. Our observations indicate (1) there are few Langerhans cells in nests of basal cell carcinoma and squamous cell carcinoma; (2) there are increased Langerhans cell densities in seborrheic keratoses, verrucous epidermal nevus, and Bowen's disease; (3) there is an expression of class II molecules on the keratinocytes and cancer cells of basal cell carcinoma, squamous cell carcinoma, Bowen's disease, seborrheic keratosis, and verrucous epidermal nevus; and (4) there is a netlike staining of the keratinocyte surface with OKM5 in the epidermal lesion of seborrheic keratosis, verrucous epidermal nevus, and Bowen's disease, as well as in the epidermis adjacent to the basal cell carcinoma and squamous cell carcinoma nests.     

A method for the rapid isolation of human epidermal Langerhans cells using immunomagnetic microspheres

Because Langerhans and indeterminate cells are the only epidermal cells that express the specific CD1a surface antigen T6, we have used immunomagnetic monodisperse polymer microspheres for positive selection of human epidermal Langerhans and indeterminate cells. Epidermal cells in suspension are successively incubated with a murine monoclonal anti-T6 antibody of the IgG1 subclass and then with magnetic beads coated with a sheep anti-mouse IgG1. Rosetted cells are obtained and then easily separated from the non-rosetted cells using a magnet. The two cell fractions are characterized by phase contrast microscopy, immunofluorescence, electron microscopy, and the skin cell-lymphocyte reaction. All the rosetted cells (1.5 to 5% of the total epidermal cells) express T6 antigen by indirect immunofluorescence and under the electron microscope possess all the ultrastructural characteristics of Langerhans cells. Moreover, the rosetted Langerhans cells remain functional: Under the electron microscope they internalize by receptor-mediated endocytosis gold labeled anti-T6 antibody, and in the skin cell-lymphocyte reaction they stimulate allogeneic lymphocytes. In contrast, the rosette depleted cell fraction is deprived of T6 positive cells and unable to stimulate allogeneic lymphocytes. The immunomagnetic depletion of epidermal cells is a simple and rapid method to isolate functional human Langerhans cells with good yield and high purity (97%). This technique should be of value in the study of the pharmacology of Langerhans cells and in the investigation of the interactions of Langerhans cells with keratinocytes or lymphocytes.