免疫磁性分离技术分离,纯化小鼠表皮郎格罕细胞

为了探讨表皮郎格罕细胞（ＬＣ）的培养方法，研究其功能特点，须将ＬＣ与表皮其它细胞相分离。我们在Ｐｅｒｃｏｌｌ密度梯度离心技术纯化ＬＣ的基础上，采用免疫磁性分离技术，取得了更为满意的结果。通过生物素－亲合素的介导，用结合大鼠抗小鼠细胞表面Ｉａ抗原的单抗的免疫磁珠吸附ＬＣ表面，经高强度磁场筛选，从而将ＬＣ从表皮细胞悬液中分离、纯化出来。结果表明，ＬＣ的纯化率超过９９％，纯化后细胞活性超过９５％，产率超     

皮肤科学基础

960439 Percoll密度梯度离心技术分离纯化豚鼠表皮Langerhans细胞/郑茂荣…//中华皮肤科杂志。-1995,28(5).-296～298 表皮Langerhans细胞(LC)虽仅占表皮细胞的2％～4％,但在皮肤免疫系统中起着重要作用。为进一步研究LC的表型、功能特点及分子生物学水平等,获得高纯化率、具有功能活性的LC很有必要。作者采用连续、间断Percoll密度梯度离心相结合的方法分离、纯化豚鼠表皮LC,结果表明LC纯化率超过60％,产率超过80％,细胞活性超过90％。自体混合白细胞应答检测证实纯化的LC仍保持功能活性。本法具有简单、快速、价廉的特点。表2参6     

高梯度磁场免疫磁珠法分离人表皮Langerhans细胞

采用鼠抗人 OKT6单克隆抗体与包被羊抗鼠 Ig G免疫磁珠的特异性结合 ,利用钢毛产生高梯度磁场 ,进行免疫磁珠法分离纯化人表皮 L C。结果表明 LC纯化率超过 99% ,细胞活性超过 95%。该方法简单、快速、价廉、纯化率高 ,不受样本量的限制 ,易在无菌下分离纯化 ,为国内首次报道     

表皮郎格罕细胞表达高亲和力IgE-Fc受体

为了进一步证实抗Ｆ　ＲＩｍＡｂ特异性地结合至郎格罕细胞（ＬＣ）表面，我们用免疫金标记技术进行了免疫电镜检查。发现５ｎｍ金颗粒不连续性地分布于ＬＣ，而非其它表皮细胞表面，从而证明ＬＣ是抗Ｆｃ　ＲＩ反应性表皮细胞。最后，为了了解ＬＣ的Ｆｃ　ＲＩ基因表达与否，进行了ＰＣＲ试验。结果显示在富集ＬＣ而非去除ＬＣ的表皮细胞悬液中存在Ｆｃ　ＲＩａ、　和　链的转录物。总之，本研究提供了直接证据证明正常人表皮ＬＣ表达Ｆ　ＲＩ。     

皮肤科学基础

980583 高梯度磁场免疫磁珠法分离人表皮Langer-hans细胞/牟贤龙（第二军医大学长海医院皮肤科）…//中国皮肤性病学杂志.-1997,11（5）.-264 Langerhans细胞（LC）在皮肤免疫系统中起着重要作用。为了深入研究LC的分子生物学特性和功能,用鼠抗人OKT6及包被羊抗鼠IgG免疫磁珠,以提高其特异结合率,在高梯度磁场中进行免疫磁珠法分离纯化人表皮LC。结果表明,LC纯化率超过99％,细胞活性超过95％。用0.3％Dispase酶分离表皮,表皮易与真皮分离,分离后的表皮结构完整。该方法简单快速、价廉、纯化率高,不受样本量的限制,易在无菌下分离纯化。表1参5 （刘辅仁）     

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。     

表皮郎格罕细胞表达高亲和力IgE-Fc受体

为了进一步证实抗Ｆ　ＲＩｍＡｂ特异性地结合至郎格罕细胞（ＬＣ）表面，我们用免疫金标记技术进行了免疫电镜检查。发现５ｎｍ金颗粒不连续性地分布于ＬＣ，而非其它表皮细胞表面，从而证明ＬＣ是抗Ｆｃ　ＲＩ反应性表皮细胞。最后，为了了解ＬＣ的Ｆｃ　ＲＩ基因表达与否，进行了ＰＣＲ试验。结果显示在富集ＬＣ而非去除ＬＣ的表皮细胞悬液中存在Ｆｃ　ＲＩａ、　和　链的转录物。总之，本研究提供了直接证据证明正常人表皮ＬＣ表达Ｆ　ＲＩ。     

长波紫外线对小鼠朗格汉斯细胞生物学活性的影响

目的　研究体外长波紫外线 (UVA)对小鼠表皮朗格汉斯细胞 (LC)生物学活性的影响。方法　观察不同剂量的UVA对BALB/c小鼠表皮LC存活率、膜相关抗原 (Ia、B7 1、B7 2、CD40 )的表达及其抗原提呈功能的影响。结果　生理剂量的UVA (<80kJ/m2 )照射 ,对LC的存活率影响不明显 ,却能增加LC膜抗原的表达 ,促进其抗原提呈功能。超生理剂量的UVA则反之。结论　生理与非生理剂量的UVA对LC会产生不同的生物学效应 ,这可能是UVA调节机体免疫功能的分子基础。     

地方性慢性砷中毒皮损内郎格罕细胞的研究

采用单克隆抗体Ｌｅｕ６间接免疫酶标法对新疆车排子地区地方性慢性砷中毒（ＥＣＡ）２５例３０份皮损内郎格罕细胞（ＬＣ）进行研究，发现２５份非肿瘤性角化症病变区表皮的角质层增厚，表皮内ＬＣ数增加，３份色素异常性皮损的表皮色素颗粒减少或消失区内ＬＣ数较多。２份恶性角化症，且份为鲍温病，其病变区内ＬＣ数目较多，而另１份鳞癌的癌巢中未见ＬＣ。认为砷角化症及色素异常的发生、发展及皮肤癌变的可能与皮肤ＬＣ有着密切的关系。     

尖锐湿疣患者郎格罕细胞观察

用ＯＫＴ６单抗及ＡＢＣ法观察了１８例尖锐湿疣患者包皮病损中郎格罕细胞（ＬＣ）的数量、分布及形态，并与正常对照组及疣旁“正常”皮肤中的ＬＣ进行了比较。结果表明，皮损表皮中ＬＣ密度明。减少，与对照组和疣旁“正常”皮肤相比，Ｐ＜０．００１，并有胞突缩短、减少或消失等改变。ＬＣ减少可能使ＨＰＶ抗原不易提是给真皮中的免疫活性细胞，以致病损长期存在，不易消退。     

Isolation and preliminary biochemical characterization of the human epidermal Langerhans cell

A method is described for the isolation of human epidermal Langerhans cells (LC). Following the disaggregation of the epidermis by gentle trypsinization, the cell suspension is incubated with the fluorescein-conjugated monoclonal antibody OKT6. Using a fluorescence-activated cell sorter, a subpopulation is selected on the basis of positive fluorescence and low forward-scatter, the latter parameter reducing contamination by LC-keratinocyte clusters. The LC averaged 1.7% of the original epidermal preparation. The purity averages 83% as judged either by OKT6 binding or by ATPase activity. Biochemical measurements indicated that the activity of lysosomal enzymes was low with the exception of alpha-mannosidase, which was about 12-fold higher than that of the keratinocyte. The activities of 3 enzymes of intermediary metabolism suggest that the utilization of glucose by the LC is considerably greater than that of the keratinocyte.     

Enrichment of unlabeled human Langerhans cells from epidermal cell suspensions by discontinuous density gradient centrifugation

In this report we introduce an alternative procedure for enrichment of human epidermal Langerhans cells (LC) from epidermal cell suspensions of normal skin. By means of discontinuous Ficoll-Metrizoate density gradient centrifugation, a fraction containing high numbers of viable, more than 80% pure LC was recovered, as judged by CD1a expression. The purity of the LC-enriched fraction appeared to be dependent on the percentage LC in the crude epidermal cell suspension. LC enriched by this method retained their accessory and antigen-presenting capacities, as determined in the Concanavalin-A induced T-cell response, in the allogeneic mixed leukocyte reaction and in the antigen-specific T-cell proliferation assay in vitro. The great advantage of this method is that it is simple and rapid and that the isolated LC are unlabeled.     

Effective enrichment of murine epidermal Langerhans cells by a modified(mismatched) panning technique.

A method for the enrichment of murine epidermal Langerhans cells (LC) is described in detail. It is based on positive selection of LC from pre-enriched fresh or cultured epidermal cell suspensions derived from ear skin by a modified panning technique. The method uses the interspecies cross-reactivities of anti-immunoglobulin antibodies: when LC in an epidermal cell suspension are labeled with mouse anti-major histocompatibility complex (MHC) class II antibodies they bind to petri dishes coated with anti-rat immunoglobulin antibodies. We therefore call this method "mismatched panning." After rinsing off non-adherent cells, the adherent LC can easily be dislodged by adding excess amounts of rat immunoglobulins, which effectively compete with the LC-bound mouse anti-MHC class II antibodies for binding to the petri dish. Using this modified panning technique, both fresh and cultured LC could be enriched up to more than 90% purity. From one ear, 2.0-3.0 x 10(4) fresh LC and 3.0-4.5 x 10(4) cultured LC could be obtained. Of all LC present in a primary, unenriched epidermal cell suspension, 40-60% were recovered when panned immediately after isolation of the epidermal cells and 50-75% when panned after 3 d of epidermal cell culture. Viability of panned LC was consistently more than 90%. Antigen presenting and T-cell-stimulating capacity of LC and responses to the cytokines granulocyte/macrophage colony-stimulating factor and tumor necrosis factor-alpha were not impaired by this panning procedure. The major advantage of this method compared to pre-existing panning techniques is the ease with which adherent LC can be dislodged from the panning dishes. Because the elution procedure is very gentle, virtually all panned LC are viable. As a consequence, good yields of highly enriched LC can be obtained in a reasonable time.     

Rapid purification of human Langerhans cells using paramagnetic microbeads

Abstract Detailed studies on the biology of Langerhans cells (LC), which account for only 1-3% of all epidermal cells, require isolation from their cutaneous symbionts. Several techniques of LC isolation have been reported, including positive enrichment with mAb coupled to immuno-magnetic beads. The disadvantage of this technique is the size of the beads (= 2-5 μm), which can interfere with subsequent phenotypic and functional analyses. This limitation prompted us to test wheter paramagnetic microbeads (15 nm) employed by the MACS? system could be used to purify LC from human skin. To isolate fresh LC (fLC), epidermal cell suspensions (EC) were stained with anti-CD la mAb and with appropriate secondary reagents conjugated to microbeads and to FITC. They were then passed over a separation column and exposed to a strong magnetic field. Thereafter both CD la-depleted and CD la-enriched cells were collected. Cultured LC (cLC) were isolated by staining 72-h cultured EC with anti-HLA-DR mAb followed by the same isolation procedure. Using this technique, we could routinely isolate viable EC that were 45-88% CD la or HLA-DR⁺ as determined by FACS. Two-color FACS analysis demonstrated the majority of MACS-purified cells to be CDla⁺/HLA-DR⁺, indicating that they were indeed LC. By transmission electron microscopy (TEM), the MACS-purified CDla⁺/HLA-DR⁺ cells showed typical ultrastructural characteristics of LC. Furthermore, MACS-purified fLC or cLC were functionally intact, because they stimulated the proliferation of alloreactive T cells in a primary, one-way, mixed epidermal cell leukocyte reaction (MECLR). We conclude that MACS-separation is an efficient and rapid method to isolate human fLC and cLC of high purity and unimpaired function.     

Comparative immunotoxicology of ultraviolet B exposure I. Effects of in vitro and in situ ultraviolet B exposure on the functional activity and morphology of Langerhans cells in the skin of different species

Ultraviolet (UV) B-induced morphological and functional changes in the skin of mice, rats and humans were investigated. Changes in the morphological structure of Langerhans cells (LC), the major antigen-presenting cells in the skin, using confocal laser scanning microscopy, were found in mouse and rat skin after in situ exposure to high doses of UVB radiation (FS40) (3–9 kJ/m²). Similar UVB doses failed to induce alterations in the morphological structure of human LC. Alterations in the function of epidermal cells (especially LC) were studied, using the mixed skin lymphocyte response (MSLR). In vitro UVB exposure of epidermal cells (EC), derived from the skin of the different species, revealed that low doses of UVB radiation impaired the stimulatory capacity of these cells dose-dependently; mouse epidermal cells were most UVB-susceptible, while human cells were least UVB susceptible. For suppression of the stimulatory capacity of EC after in situ UVB exposure of skin tissue, higher doses of UVB radiation than the in vitro UVB exposure were needed in all species tested. Also in this in situ set-up mouse epidermal cells were most UVB-susceptible, and human epidermal cells were least UVB-susceptible. The magnitude of differences in susceptibility for UVB-induced changes in the stimulatory capacity of EC after in situ and after in vitro exposure experiments was similar. Firstly, it may be concluded that UVB impairs the functional activity of LC at a lower dose than that which alters the morphology of these cells. Secondly, it is clear that epidermal cells, especially LC, from the skin of rodents are more susceptible to UVB than epidermal cells derived from human skin. It is important to account for these differences in susceptibility when data on the effects of UVB radiation on the immune system in rodents are extrapolated to humans.     

Granulocyte macrophage--colony-stimulating factor (GM-CSF) decreases CD1a expression by human Langerhans cells and increases proliferation in the mixed epidermal cell-lymphocyte reaction (MELR).

Langerhans cells (LC) undergo a variety of phenotypic and functional changes in vitro. To determine the effects of granulocyte macrophage--colony-stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-alpha), and interleukin 1-alpha (IL-1) on LC phenotype in vitro, epidermal cell suspensions were enriched for LC by density-gradient centrifugation and cultured in the presence of 10 ng/ml of these cytokines. The percentage of cells expressing the surface protein CD1a was determined by flow cytometry. This percentage typically dropped after 48 h culture in both control and cytokine-treated medium to less than half that of the starting value. By the fifth day, the percentage of cells expressing CD1a in TNF-alpha and IL-1--treated cultures was still near half of the starting value, slightly above that of control cultures. Treatment with GM-CSF caused large and consistent decreases in the percentage of epidermal cells expressing CD1a. Cell viability in each of the three cytokine-treated cultures was identical to the control cultures, with essentially all cells having died by the sixth day after isolation. To determine the functional effects of these cytokines, the cytokine-containing medium was replaced after 72 h with medium containing purified allogeneic T cells and proliferation measured. Preliminary experiments showed no increased proliferation induced by IL-1 or TNF-alpha--treated epidermal cells. GM-CSF-treated epidermal cells induced 2-3 times more T-cell proliferation than epidermal cells cultured without additional cytokines. We conclude that GM-CSF, a cytokine known to be produced by keratinocytes in vitro, decreases CD1a expression by human LC and increases their ability to stimulate proliferation by allogeneic T cells.     

Enrichment of human epidermal Langerhans cells by attachment to erythrocyte monolayers

The present investigation introduces a method for purification of human epidermal Langerhans cells (LC). The method is based on the attachment of LC to IgG-coated sheep erythrocyte monolayers via their Fc receptors. To optimize the enrichment assay, several variables were tested. The best results were obtained when epidermal cells were centrifuged against erythrocyte monolayers; the purification procedure was performed at 4 degrees C in the presence of 5% fetal calf serum, using about 6 X 10(6) epidermal cells per erythrocyte plate (diameter 5 cm). The average purity of the recovered LC was 80.9% and LC-depleted fractions contained an average of 0.5% DR-positive cells. LC were able to enhance significantly leukoagglutinin- and purified protein derivative-induced T lymphocyte proliferation and leukocyte migration inhibitory factor production.     

Adhesion molecules CD11a, CD18, and ICAM-1 on human epidermal Langerhans cells serve a functional role in the activation of alloreactive T cells.

Binding of antigen-presenting cells (APC) to T cells via adhesion molecules is thought to deliver accessory signals that are required for efficient T-cell activation. To determine whether Langerhans cells (LC) express relevant adhesion molecules on their surfaces, we employed two-color immunofluorescence. Human epidermal cells (EC), Ficoll-enriched for LC (greater than 10%), were incubated with monoclonal antibodies (MoAb) specific for the adhesion molecules CD11a (LFA-1 alpha), CD18 (LFA-1 beta), or ICAM-1; staining was evaluated by fluorescence microscopy. After 12 h of culture only HLA-DR+ cells (LC) expressed CD11a, CD18, and ICAM-1. As a test for the functional relevance of such adhesion molecule expression, we examined the capacity of the above MoAb to block LC stimulation of alloreactive T cells: EC were co-cultured with allogeneic peripheral blood mononuclear leukocytes (PBML) for 5 d in the presence or absence of MoAb; proliferation was measured by [3H]-thymidine uptake. MoAb against CD11a, CD18, or ICAM-1 reduced the allostimulatory capacity of LC by greater than 70%; combinations of these MoAb reduced proliferation even more (90%). We conclude that interaction of adhesion molecules on LC with ligands on T cells is required for optimal allo-antigen-dependent T-cell activation, perhaps by delivering accessory signals.     

The steady-state turnover of murine epidermal Langerhans cells

We have investigated the steady-state turnover of murine epidermal Langerhans cells (LCs) using an X-irradiation model, 3H-thymidine autoradiography and cultured epidermal sheet explants, and by assessing the LC population in normal mice. The LC density after whole-body irradiation without any cutaneous shielding was not significantly different from that in skin shielded during whole-body irradiation (P > 0.05), indicating that the additional irradiation to the skin did not contribute to a decrease in LC density. In both instances, the LC number gradually decreased in a linear fashion. The results indicate that epidermal LCs continuously leave the epidermis and are continually replaced by circulating precursor cells from the bone marrow at a steady rate. Autoradiographic studies after a pulse injection of 3H-thymidine showed a labelling index of 0.013%, indicating that local mitosis is not an important contributor to the maintenance of the epidermal LC population. Although local X-irradiation resulted in temporary reduction of LC density, epidermal sheet explant culture obtained immediately after local X-irradiation showed no difference in LC density as compared with control unirradiated skin, indicating that the decrease in LC density was not due to significant LC destruction. From these data, we calculated that the half-life of murine LCs in the epidermis is approximately 9 days.     

CD70-CD27 interaction augments CD8+ T-cell activation by human epidermal Langerhans cells

Human cutaneous dendritic cells (DCs) from epidermal and dermal compartments exhibit functional differences in their induction of CD4+ T-cell and humoral immune responses; however, differences in the regulation of memory CD8+ T-cell responses by human skin DCs remain poorly characterized. We tested the capacity of human Langerhans cells (LCs) and dermal dendritic cells (DDCs) to induce antigen-specific cytokine production and proliferation of memory CD8+ cells. Although tumor necrosis factor-α-matured human DCs from both epidermal and dermal compartments showed efficient potential to activate CD8+ cells, LCs were constitutively more efficient than DDCs in cross-presenting CD8+ epitopes, as well as direct presentation of viral antigen to Epstein-Barr virus-specific CD8+ T cells. LCs showed greater expression of CD70, and blockade of CD70-CD27 signaling demonstrated that superiority of CD8+ activation by epidermal LC is CD70 dependent. This CD70-related activation of CD8+ cells by LCs denotes a central role of LCs in CD8+ immunity in skin, and suggests that regulation of LC CD70 expression is important in enhancing immunity against cutaneous epithelial pathogens and cancer.