联用密度梯度离心与免疫磁珠法分离纯化人表皮朗格汉斯细胞

目的:探讨获取较高纯度和活性的人表皮朗格汉斯细胞(LC)的实验方法。方法:联合采用密度梯度离心和免疫磁珠的方法分离纯化人表皮LC,将分选后的细胞用结合有异硫氰酸荧光素(FITC)的鼠抗人CD1a单抗标记,经流式细胞仪检测LC的纯化率,采用0.4%锥虫蓝染色检测细胞的活性。结果:分选所得LC纯化率为84.48%,锥虫蓝染色显示细胞活性>95%。结论:联用密度梯度离心和免疫磁珠法,可获得较高纯度、有活性的LC细胞,且具有操作简单、易于无菌条件下分离纯化的优点。     

In situ characterization of cellular infiltrates in lupus vulgaris indicates lesional T-cell activation.

Skin biopsy specimens from nine patients with lupus vulgaris were examined in situ by means of monoclonal antibodies directed against phenotypes of lymphocyte subsets, Langerhans cells, HLA-DR antigens, and interleukin 2 receptor. The epidermis showed prominent changes, including intense expression of HLA-DR on keratinocytes, increase in epidermal cell layers, moderate to high Langerhans cell hyperplasia, and infiltration by CD3+ pan-T cells as well as CD8+ (cytotoxic/suppressor) and CD4+ (helper/inducer) T cells. The predominant lymphocyte in the dermal granulomas was the activated CD3+ T cell, expressing major histocompatibility complex class II antigens and interleukin 2 receptor. CD4+ and CD8+ cells were randomly distributed among the epithelioid cells, which showed intense staining for major histocompatibility complex class II antigens. In all except two patients, the CD4+ population was greater than that of the CD8+ cells. CD1+ Langerhans cells were scattered in moderate numbers in the dermal granulomas. Acid-fast bacilli were conspicuously absent in the biopsy specimens. These features suggest that T-cell activation and Langerhans cell hyperplasia are prominent features of dermal tuberculosis.     

Studies on human epidermal Langerhans cells

Human epidermis from nickel sensitive patients was separated from dermis by means of a suction blister device and dissociated with trypsin. The epidermal cell suspensions obtained contained 3–5% Langerhans cells as judged by immunofluorescence staining of the cells with a rabbit anti-DR anti-serum. The epidermal cells were co-cultured with purified autologous T lymphocytes with or without nickel sulphate. A strong proliferative T lymphocyte response to nickel sulphate was obtained provided epidermal cells were also present. Pretreatment of the epidermal cells with anti-DR antiserum abolished or greatly reduced the response. These data indicate that epidermal cells are able to present nickel sulphate to T lymphocytes in an immunogenic way. Since the responsible cells were DR antigen positive, it is highly probable that the cells responsible for these functions are the Langerhans cells.     

Changes in Lymphocyte and Langerhans Cell Populations in Allergic and Irritant Contact Dermatitis

We observed in situ changes in lymphocyte subpopulations and Langerhans cells during allergic and irritant contact dermatitis using immunohistochemical staining methods with monoclonal antibodies to cell surface antigens. In both types of contact dermatitis, there was a perivascular infiltrate of T lymphocytes, with helper/inducer T cells predominating. B cells were absent, and natural killer cells were absent or sparse. During the course of allergic contact dermatitis, Langerhans cells showed a striking sequential change in location, with the cells first in the epidermis, then perivascularly in the dermis (days 1-14), and returning to the epidermis (days 14-21). In irritant contact dermatitis, the Langerhans cells were initially identified in the epidermis and then appeared diffusely in the dermis (days 1-2). The numbers in the dermis then decreased abruptly (day 4). They were again identified in normal numbers in the epidermis (day 21). The response of Langerhans cells appears to be different between allergic and irritant contact dermatitis.     

Thymopoietin-like substance in human skin

A heterologous antithymopoietin (anti-TP) antibody was used to determine whether a TP-like molecule is present in the epidermis, since such factors have been postulated to play a part in known T cell-epidermal cell interaction. Examination of cytocentrifuge smears of freshly separated human epidermal cells stained by indirect immunofluorescence revealed that 8-14% of these cells possessed cytoplasmic reactivity with the anti-TP antibody. Similarly, 2-5% of human epidermal cells, maintained in tissue culture for 2-8 weeks, showed cytoplasmic staining with the anti-TP antibody. Double-labeling immunofluorescence studies, with the anti-TP antibody and a monoclonal antibody specifically reactive with Langerhans cells (OKT6), demonstrated that cells possessing this TP-like substance were not Langerhans cells. In situ studies of 4-microns frozen sections of normal human skin indicated that the cell population which possesses the TP-like substance is the basal layer of keratincoytes in the epidermis.     

Reactivity of epidermal Langerhans cells to a histochemical method for demonstration of beta-glucuronidase

Sheets or sections of mouse epidermis reacted by a histochemical method for the enzyme beta-glucuronidase display a subpopulation of dendritic cells which correspond in number and spacing to Langerhans cells demonstrated by reactivity for ATPase or Ia antigens. A similar staining pattern is seen in rat, rabbit, and guinea pig epidermis. In rhesus monkey and human skin, Langerhans cells appear to be reactive for beta-glucuronidase but, as keratinocytes are also reactive, Langerhans cells are not readily identifiable by this method. The thermal stability of beta-glucuronidase differs between strains of mice. Langerhans cells of Balb/C and C3H strains can thus be distinguished by appropriate pretreatment before incubation, a method of potential value for experimental investigations of the origin of Langerhans cells.     

The repopulation of murine Langerhans cells after depletion by mild heat injury

We have developed a model of focal Langerhans cell depletion by mild heat injury and used it to investigate the mechanisms of Langerhans cell repopulation in the injured epidermis. The possibility whether repopulation occurred by recruitment of precursor cells from the circulation or dermis or, alternatively, by migration from the surrounding normal epidermis into the injured area was considered. Repopulation was studied by evaluating the pattern of Langerhans cell reappearance and calculating the rate of change in the density. Heat injury followed by whole-body irradiation with shielding of the injured skin was used to assess repopulation in the absence of bone marrow precursors. Using tritiated thymidine autoradiography, we also investigated whether the newly arrived Langerhans cells (be they from circulating precursors or surrounding normal epidermis) actually divide. The results showed that heat injury completely eliminated the Langerhans cells within the area delineated by the injury. Two hours after injury, the Langerhans cells were fragmented and 2 days later, they could not be detected. Regeneration of the epidermis occurred 2 days after injury and Langerhans cells reappeared scattered somewhat sparsely in the centre of the lesion on day 3. These cells were small and slender, bearing one or two short dendrites. As the dendrites increased in number and in length, the cells became similar morphologically and phenotypically to normal Langerhans cells. The rate of repopulation increased dramatically between days 5 and 7 and reached normal density on day 11. The pattern of Langerhans cell repopulation in the injured area and the lack of repopulation in the irradiated animals indicated that repopulation occurs by immigration of precursors from the circulation or dermis. There was no indication of migration of Langerhans cells from surrounding normal epidermis. Lastly, the newly arrived Langerhans cells failed to divide at the site of injury.     

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。     

Location of HLA-A,B,C antigens in dendritic cells of normal human skin: an immunoelectron microscopic study

The distribution of the major histocompatibility antigens HLA-A,B,C, (HLA) on dendritic cells of normal human skin was studied by immunoelectron microscopy and a 4-step immunoperoxidase technique utilizing monoclonal antibodies. Light microscopy revealed peripheral staining for HLA of epidermal and pilar infundibular keratinocytes. In the epidermis, the staining was present from the basal layer to the upper stratum spinosum. In the follicles below the level of the infundibulum, HLA was detected only on rare intraepithelial dendritic cells. These dendritic cells could not be identified in the epidermis due to the HLA staining of the surrounding keratinocytes. Similar cells stained diffusely with anti-T6 antibody; the keratinocytes did not. Immunoelectron microscopy demonstrated: (1) the presence of HLA staining of keratinocyte membranes from the stratum basalis to the level of the upper stratum spinosum and in the pilar infundibulum, (2) the possible absence of HLA on melanocytes, (3) the presence of focal HLA staining of the membranes of epidermal and follicular dendritic cells that contained Birbeck granules and were, therefore, Langerhans cells, (4) dendritic mononuclear cells within the follicular epithelium, which although devoid of Birbeck granules, exhibited similar reactivity with anti-HLA antibody. These findings suggest that HLA antigens are present on the membranes of Langerhans cells, but are not demonstrable on melanocytes in normal human skin.     

他克莫司对朗格汉斯细胞迁移能力的影响

目的：观察他克莫司对朗格汉斯细胞（LC）迁移能力的影响,为明确他克莫司在治疗疾病中的作用途径打下基础。方法：在不同时间段内将不同浓度的他克莫司腹腔注射C5UBL／6小鼠,再用异硫氰酸荧光素（FITC）刺激小鼠耳背部皮肤,12h后通过流式细胞仪检测小鼠耳背部淋巴结内摄取抗原的LC数量。通过免疫组化法检测抗原致敏处LC的数量。结果：他克莫司腹腔注射过的小鼠耳背皮肤受到FITC刺激后,其皮肤局部摄取抗原的LC迁移至局部淋巴结的数量明显减少,尤其在FITC刺激前12h注射他克莫司的小鼠皮肤局部LC的迁移能力受到显著抑制,在注射高剂量他克莫司组与低剂量且之间存在明显差异。而同时他克莫司腹腔注射过的小鼠,其耳背部受到FITC刺激后的皮肤局部LC的数量高于未注射组。结论：他克莫司作为一种免疫抑制剂,其发挥治疗作用途径有可能通过抑制LC的迁移能力,从而上或减弱了某些免疫反应。     

Control of the differentiation state and function of human epidermal Langerhans cells by cytokines in vitro

Langerhans cells can originate in vitro from immature precursors stimulated with granulocyte macrophage-colony-stimulating factor (GM-CSF), tumour necrosis factor (TNF)-alpha and stem cell factor (SCF). We asked whether these cytokines also control the differentiation state of Langerhans cells within the epidermis and upon leaving this tissue. We harvested sheets of human epidermis by controlled dispase hydrolysis of keratomes, cultured them in RPMI and 10% fetal calf serum for 48 h and analysed the sheets and the cells migrated spontaneously into the medium, most of which were Langerhans cells containing Birbeck granules. By flow cytometry, the intensity of CD1a expression was reduced quite evenly among Langerhans cells migrated from sheets within 48 h. The cells in the sheets underwent loss of dendrites, with a significant reduction in the cell perimeter that was prevented by GM-CSF and TNF-alpha together. Either of these cytokines induced expression of CD18 by cells in the sheets and those in the medium. Moreover, TNF-alpha induced expression of CD54 by cells in the medium, but not by those retained in the sheets, whereas human SCF induced, dose dependently, expression of CD54 by cells in the sheets, but not from those in the medium. The proliferation of allogeneic lymphocytes was much higher when stimulating Langerhans cells were harvested from cultures with any cytokine, rather than from cultures without cytokines. We conclude the following: (i) GM-CSF and TNF-alpha help to maintain full differentiation of Langerhans cells within the epidermis; (ii) cytokine influence on Langerhans cells adhesiveness is in part context dependent; and (iii) pretreatment with cytokines influences positively the number or accessory activity of Langerhans cells on lymphocytes during subsequent mixed leucocyte reaction.     

Human epidermal Langerhans cells express the tight junction protein claudin-1 and are present in human genetic claudin-1 deficiency (NISCH syndrome)

Abstract:  Claudin-1 (CLDN1) is a structural tight junction (TJ) protein and is expressed in differentiating keratinocytes and Langerhans cells in the epidermis. Our objective was to identify immunoreactive CLDN1 in human epidermal Langerhans cells and to examine the pattern of epidermal Langerhans cells in genetic human CLDN1 deficiency [neonatal ichthyosis, sclerosing cholangitis (NISCH) syndrome]. Epidermal cells from healthy human skin labelled with CLDN1-specific antibodies were analysed by confocal laser immunofluorescence microscopy and flow cytometry. Skin biopsy sections of two patients with NISCH syndrome were stained with an antibody to CD1a expressed on epidermal Langerhans cells. Epidermal Langerhans cells and a subpopulation of keratinocytes from healthy skin were positive for CLDN1. The gross number and distribution of epidermal Langerhans cells of two patients with molecularly confirmed NISCH syndrome, however, was not grossly altered. Therefore, CLDN1 is unlikely to play a critical role in migration of Langerhans cells (or their precursors) to the epidermis or their positioning within the epidermis. Our findings do not exclude a role of this TJ molecule once Langerhans cells have left the epidermis for draining lymph nodes.     

Ia antigens on indeterminate cells of the epidermis: immunoelectronmicroscpic studies of surface antigens

An antiserum against human B-lymphoblastoid cell membrane alloantigens (Ia-like antigens) was used to study the presence of such antigens on dendritic cells in human epidermis. Only Langerhans cells and the majority (85%) of so-called indeterminate cells were positively stained, as shown by immuno-electron microscopy. Fifteen percent of the indeterminate cells were negative and were considered to be immature melanocytes. A relationship exists between the indeterminate cell and the Langerhans cell. A proposal is made concerning emigration of Langerhans cells in response to haptenic stimulation, and the immigration of indeterminate cells to restore the status quo.     

Selective uptake of contact allergens by the Langerhans cell.

Using histochemical staining techniques, it has been possible to demonstrate the selective uptake of ten different contact allergens by the Langerhans cells. It is postulated that the Langerhans cells form a reticuloepithelial system that clears the epidermis of foreign chemicals. The Langerhans cell is viewed as the site of hapten binding and antigen formation and hence the central target cell in immune contact dermatitis reactions.     

结合珠蛋白在正常人皮肤中的定位表达

目的 探讨正常人皮肤中结合珠蛋白及其mRNA的表达在皮肤中的作用及其与朗格汉斯细胞的关系。方法 采用过氧化物酶法及荧光双标记法和原位杂交技术检测30例正常人皮肤中结合珠蛋白的表达。结果 角质形成细胞、毛囊上皮、皮脂腺导管上皮细胞、腺上皮及小汗腺细胞结合珠蛋白mRNA呈弱阳性至中度阳性表达,真皮成纤维细胞和血管内皮细胞中均未见阳性表达,而在蛋白质水平,只见到一些树突细胞呈阳性表达,免疫荧光双标记检测证实为朗格汉斯细胞。结论 皮肤是结合珠蛋白肝外表达的又一器官,角质形成细胞等上皮来源的细胞是结合珠蛋白mRNA表达的主要细胞;正常皮肤中只见到表皮中下部、毛囊、皮脂腺导管部位的朗格汉斯细胞胞浆中含有结合珠蛋白;角质形成细胞中结合珠蛋白mRNA的表达可能与朗格汉斯细胞胞浆中的结合珠蛋白有关;结合珠蛋白可能参与皮肤中的一些免疫反应过程。     

Antigen presentation and T-cell activation in epidermodysplasia verruciformis

Aberrant immune responses may play a role in the susceptibility of patients with epidermodysplasia verruciformis to human papilloma virus (HPV). We examined the stimulatory capacity of antigen-presenting cells from HPV-infected skin and peripheral blood T-cell responses of patients with epidermodysplasia verruciformis. The percentage of Langerhans cells in relation to total epidermal cells in suspension was slightly reduced in HPV-infected lesions, relative to apparently clinically uninfected epidermis. In addition, the morphologic appearance of Langerhans cells was altered in lesional epidermal sheets. Despite these abnormalities, Langerhans cells were functionally intact in their capacity to present alloantigens to T cells and, in fact, the epidermis of HPV-infected lesions demonstrated enhanced antigen-presenting activity in three of four patients tested. The antigen-presenting activity was entirely abrogated by removal of Langerhans cells and was not associated with increased activity of cytokines with stimulatory activity for the thymocyte co-stimulation assay. Although epidermodysplasia verruciformis T cells were unresponsive to autologous HPV-infected epidermis, they responded well to mitogens, allogeneic mononuclear leukocytes, and allogeneic epidermal cells. Epidermodysplasia verruciformis T cells were inhibited in their capacity to respond to allogeneic epidermal cells when they were simultaneously exposed to autologous epidermal cells from HPV-infected lesional epidermis, but not to normal-appearing epidermis. Thus, although Langerhans cell activity is intact in epidermodysplasia verruciformis, these individuals fail to respond to autologous papillomas, which may, at least in part, be explained by an interaction between papillomal epidermal cells and autologous T cells that results in an inhibited response.     

Immunophenotypic analysis of normal human dendritic cells isolated from epidermis and dermis

Background The skin immune system comprises two types of dendritic cells, i.e. CD1a-positive Langerhans cells in the epidermis and CD36-positive dendritic macrophages in the dermis. Dendritic cells can migrate from skin explants into a culture medium.
Methods We have examined the morphology and immunophenotype of the dendritic cells migrating from epidermal and dermal sheets in vitro. The epidermis and dermis of keratomes of normal human skin were separated with dispase and cultured for 72 h. At this time, the non-adherent cells in the medium were removed, enriched on a metrizamide or Lymphoprep gradient, counted, prepared by cytospin, and labeled for CD1a, CD36, and HLADr.
Results Cells migrating from the epidermis and dermis show many thin projections or a few veils from the cell surface. Approximately four times more cells migrate from epidermal than dermal sheets from the same keratome.
Conclusions Using methods to separate the epidermis from the dermis, both CD1a-positive Langerhans cells and CD36-positive dendritic macrophages can be obtained from both tissues, although in different numbers.     

A strikingly constant ratio exists between Langerhans cells and other epidermal cells in human skin. A stereologic study using the optical disector method and the confocal laser scanning microscope

Langerhans cells play an important part in the immune surveillance of the human epidermis. Therefore, a certain distribution and numerical relationship to other epidermal cells can be expected. To quantify epidermal Langerhans cells population extensive studies have been performed using two-dimensional quantification methods on vertical sections or epidermal sheet preparations. Whereas methods using vertical sections were complicated considerably by the sampling procedure, the dendritic shape, and the suprabasal, nonrandom distribution of Langerhans cells, epidermal sheet preparations have their limitations regarding the numerical relationship of Langerhans cells to total epidermal cells and the epidermal morphology as such. In order to improve the validity of data the three-dimensional dissector method combined with confocal laser scanning microscopy has been applied to quantify the number of Langerhans cells and other epidermal cell nuclei per volume unit in cryosections of 24 punch biopsies of normal breast skin of eight women. Furthermore, the ratio of Langerhans cells to other epidermal cells, their number per biopsy, and per skin surface area were calculated. To minimize the bias by shrinkage the reference volume was estimated using Cavalieri's principle. A constant ratio of one Langerhans cells to 53 other epidermal cells was identified in breast skin (interindividual correlation coefficient: 0.952, p < 0.0001). Thus, Langerhans cells represent 1.86% of all epidermal cells; however, a wide interindividual range was found for the number of Langerhans cells per mm2 (912-1806; mean +/- SD 1394 +/- 321) and other epidermal cells per mm2 (47,315-104,588; mean +/- SD 73,952 +/- 19,426). This explains the conflicting results achieved by conventional morphometric assessments relating cell numbers to skin surface area, ignoring the varying thickness of the epidermis. The surprisingly constant relationship of Langerhans cells to other epidermal cells stresses the hypothesis of an epidermal Langerhans cells unit where one Langerhans cells seems to be responsible for the immune surveillance of 53 epidermal cells.     

Densities,distribution and phenotypic expression of T cells in human fetal skin

T cells are present in normal adult human skin, but their occurrence in fetal skin is unknown. T cell and Langerhans cell (LC) populations were studied using single or double immunohistochemical staining on cryostat-section. Skin samples taken from different body regions of 17 fetuses ranging from 18 to 30 weeks estimated gestational-age (w-EGA), were examined. In all specimens but one, we did not find any epidermal T cell. In contrast, dermal CD3⁺ T cells occurred at all w-EGA. The density of these cells increased with increasing age. Double staining showed that CD3⁺ T cells were predominantly CD4⁺/CD45RA⁺. On the other hand, LC, as assessed by CD1a expression, was evenly distributed within the interfollicular epidermis and papillary dermis at all gestational ages. Analysis of T cell and LC density in different body regions did not show significant topographic differences. We suggest that lack of epidermal T cells, although the LC network was fully represented, might reflect the scarce opportunity of fetal LC to contact foreign antigens in utero.     

Differentiation-associated localization of small prolne-rich rotein in normal and diseased human skin

Summary The expression of SPRR (small proline-rich protein) was investigated in normal human skin and in diseased skin from patients with psoriasis, squamous cell carcinoma, basal cell epithelioma. Naevus pigmentosus, ichthyosis vulgaris and several inflammatory skin diseases, by immunohistochemical staining. A polyclonal antibody was raised against a synthetic peptide for a C-terminal common region for SPRR l and SPRR 3. In immunoblot analysis, a positive band of 18kDa was detected, which showed the presence of SPRR l in human epidermal keratinocytes. In normal epidermis, positive staining for SPRK was observed in keratinocytes in the granular layer and the uppermost or two spinous cell layers, with no staining of the other spinous or basal layers. The staining was obvious at the cell periphery, weak at the cytoplasm, and absent in the nucleus. Staining was observed in several outer layers of the follicular infundibulum to the isthmus. No staining was detected in the inner root sheath of the hair follicles, hair matrix, sebaceous gland, eccrine gland, eccrine duct, melanocytes. Langerhans cells or fibroblasts. The arrectores pilorum, striated muscles, muscle layers of vessels, and myoepithelia of eccrine gland, were weakly stained. In psoriatic skin, stained keratinocytes were distributed in the spinous cell layers except for the basal layer, in ichthyosis vulgaris. SPRR was barely expressed in the uppermost living cell layers of the epidermis in epidermolytic hyperkeratosis. degenerated squamous cells widely expressed SPRR. In Darier's disease, dyskeratolic cells were clearly stained. In squamous cell carcinoma, staining was observed in keratotic cells around horny pearls. In basal cell epithelioma, naevus pigmentosus, and malignant melanoma, the tumour cells or naevus cells were not stained. The distribution of SPRR was similar to that of involucrin in normal and several diseased skin, except for ichthyosis vulgaris. We conclude that SPRR is expressed in close association with epidermal differentiation in normal skin and skin diseases. The alteration of the expression of the proteins correlated to terminal differentiation, and differs from disease to disease.