Differentiation of Langerhans Cells from Interdigitating Cells Using CD1a and S-100 Protein Antibodies

The present study shows that Langerhans cells can be differentiated from Interdigitating cells at the light microscopic level. Superficial lymph nodes and skin taken from necropsies and the lymph nodes of dermatopathic lymphadenopathy (DPL) were used for this experiment. Sections of lymph node and skin were embedded using the acetone, methyl benzoate and xylene (AMeX) method and dendritic cells were immunostained with anti S-100 protein antibody (S-100, and OKT-6 (CD1a) using the restaining method. Langerhans cells in the skin were positive for both CD1a and S-100. Dendritic cells positive for both CD1a and S-100, and dendritic cells positive for S-100, but not for CD1a were observed in superficial lymph nodes. In normal superficial lymph nodes, there were more interdigitating cells than Langerhans cells. The majority of the dendritic cells in the DPL were Langerhans cells. We conclude that the S-100 and CD1a positive cells are Langerhans cells, and the S-100 positive-CD1a negative cells are interdigitating cells.     

Langerhans cells in the lymph node: mirror section and immunoelectron microscopic studies

Cells immunostained with antibodies against both OKT-6 and S-100 protein were observed only in superficial and hilar lymph nodes draining tissues with predominantly squamous epithelia. In contrast, in mesenteric lymph nodes and the spleen, only S-100 protein-positive, but OKT-6-negative cells were found. We suspect that the S-100 and OKT-6-positive cells might be Langerhans cells (LC) and the S-100-positive, OKT-6-negative cells, interdigitating reticulum cells (IDC). We further postulate that the LC in superficial and hilar lymph nodes might migrate from squamous epithelia, with which contact is required for the formation of Birbeck granules.     

Langerhans cells in the lymph node: mirror section and immunoelectron microscopic studies.

Cells immunostained with antibodies against both OKT-6 and S-100 protein were observed only in superficial and hilar lymph nodes draining tissues with predominantly squamous epithelia. In contrast, in mesenteric lymph nodes and the spleen, only S-100 protein-positive, but OKT-6-negative cells were found. We suspect that the S-100 and OKT-6-positive cells might be Langerhans cells (LC) and the S-100-positive, OKT-6-negative cells, interdigitating reticulum cells (IDC). We further postulate that the LC in superficial and hilar lymph nodes might migrate from squamous epithelia, with which contact is required for the formation of Birbeck granules.     

Immunoreactivity for S-100 protein in dendritic and in lymphocyte-like cells in human lymphoid tissues

S-100 protein is an immunohistochemical marker for a subset of dendritic cells, the interdigitating reticulum cells (IDRCs), which are mainly located in T-dependent areas of lymphoid tissues. In the present study we have investigated the distribution of S-100-positive cells in lymph nodes, spleen, thymus and peripheral blood of normal subjects. Immunoreactivity for S-100 protein was demonstrated in large cells with dendritic morphology and in small lymphocyte-like cells present in the lymph node paracortex, thymic medulla, splenic periarterial lymphatic sheaths (PALS) and in peripheral blood. S-100-positive lymphocyte-like cells were frequently detected around high endothelial venules (HEV) and were present in numbers comparable to those of S-100-positive IDRCs. Immunoelectron microscopy confirmed the existence of positive cells with lymphoid morphology and revealed that the intracellular distribution of the immunoreaction product was similar in lymphoid and dendritic cells. Further characterization of S-100-positive cells demonstrated that both lymphoid and dendritic cells were unreactive with a large panel of monocytic and macrophage markers.     

Migration of Murine Epidermal Langerhans Cells to Regional Lymph Nodes: Engagement of Major Histocompatibility Complex Class II Antigens Induces Migration of Langerhans Cells

Langerhans cells are resident dendritic cells in the epidermis. Once they are loaded with epicutaneously-delivered antigens, they leave the epidermis and migrate to the regional lymph nodes where they initiate primary T cell responses as antigen-presenting cells. However, the stimulus that initiates such migration remains unknown. Because major histocompatibility complex class II (Ia) antigens on B lymphocytes or monocytic cells have been shown to function as signal transducers, we evaluated the effect of the engagement of Ia antigens on the migration of murine epidermal Langerhans cells. The intradermal injection of an anti-Ia monoclonal antibody (mAb) reduced the density of Langerhans cells in epidermis and produced a dose- and time-dependent increase in the frequency of cells reactive with NLDC145 (Langerhans cell- and dendritic cell-specific mAb) within the regional lymph nodes. Injection of a control mAb had no effect. The NLDC145+ cells that were induced to accumulate in the regional lymph nodes were Ia+, large dendritic cells, some of which were positive for both NLDC145 and F4/80, a phenotype corresponding to that of murine epidermal Langerhans cells. Thus, the engagement of Ia antigens on Langerhans cells by mAb induces the migration of Langerhans cells from the epidermis to the regional lymph nodes. Analysis of these changes in Langerhans cells in vitro may help to reveal the biochemical sequence of events involved in the activation and differentiation of Langerhans cells.     

CD44 Variant Isoforms are Essential for the Function of Epidermal Langerhans Cells and Dendritic Cells

Upon antigen encounter epidermal Langerhans cells (LC) and dendritic cells (DC) emigrate from peripheral organs and invade lymph nodes through the afferent lymphatic vessels and then assemble in the paracortical T cell zone and present antigen to T lymphocytes. Part of this process is mimicked by metastasizing tumor cells. Since splice variants of CD44 promote metastasis to lymph nodes we explored the expression of CD44 proteins on migrating LC and DC. We show that following antigen contact, LC and DC upregulate pan CD44 epitopes and epitopes encoded by variant exons v4, v5, v6 and v9. Antibodies against CD44 epitopes arrest LC in the epidermis, prevent the binding of activated LC and DC to the T cell zones of lymph nodes, and severely inhibit their capacity to induce a delayed type hypersensitivity reaction to a skin hapten in vivo. Our results demonstrate that CD44 splice variant expression is obligatory for the migration and function of LC and DC.     

Significance of S-100 protein immunostaining in the immunohistological analysis of normal and neoplastic lymphoid tissues--an appraisal

S-100 protein is a heterogeneous fraction of dimeric polypeptides (alpha and beta subunits) that can exist in different combination forms within the various tissues. Concerning the S-100 protein immunodetection within lymphoid tissue, the heterogeneity of the S-100 antigen, the tissue quality (frozen or paraffin-embedded after treatment with different fixatives) and the treatment of the tissue with different immunostaining methods and antibodies of different nature, all make for inconsistent results obtained in the immunohistological studies reported in the literature. Most of the S-100-positive cells of the lymphoreticular system are dendritic cells involved in the immune response (interdigitating reticulum cells, Langerhans cells, and follicular dendritic reticulum cells), other S-100-positive cells belonging to the mononuclear/phagocytic system. S-100 protein immunostaining may be used as a helpful immunohistological diagnostic clue to certain malignancies of the immune system (follicular center cell lymphomas) on the basis of their specifically related dendritic cell microenvironment. In addition to monoclonal antibodies for the immunophenotypic characterization of dendritic cells and macrophages and to enzyme reactions, the combined use of anti-S-100 antibodies specific for each of the S-100 protein subunits, tested with sensitive procedures, would be a very useful tool in the attempt to classify the proliferative disorders of dendritic cells and macrophages.     

Coexpression of CD1a, langerin and Birbeck's granules in Langerhans cell histiocytoses (LCH) in children: ultrastructural and immunocytochemical studies

Langerhans cell histiocytoses (LCH) represent rare diseases of unclear etiology and pathogenesis. Most of the cases include children, 1 to 15 years of age, and various organs are involved (bones, skin, liver, lymph nodes, bone marrow and other). The diagnosis of LCH used to be established by biopsy of the inflamed tissue and demonstration of expression of markers specific for Langerhans cells: CD1a and langerin. The diagnosis can be ultimately confirmed by demonstration of Birbeck's granules in the electron microscopy. The present study was aimed at immunocytochemical demonstration, in the examined LCH material (skin, bones, lymph nodes), of the specific antigen expression and at comparing it with the presence of Birbeck's granules. In the examined 11 cases co-expression of CD1a with langerin and with the presence of Birbeck's granules was noted. Also in all examined biopsies the expression of S-100 protein on inflammatory cells was found. The results corroborate the usefulness of immunocytochemical studies on CD 1 a and langerin expression in diagnosis of LCH.     

Abundant dendritic cells express HLA-DR in pleomorphic adenomas

Most pleomorphic adenomas were found to contain abundant dendritic cells (DC) with major histocompatibility complex (MHC) class II (HLA-DR) expression. Their immunohistochemical staining features were suggestive of dendritic histiocytic cells. Extensive phenotypic characterization by two-colour immunofluorescence staining for various cell markers was performed. The DC expressed both HLA class I and II determinants, vimentin, S-100 protein, and various monocyte-related markers (10G11, 3D10, 7G5 or CD11a, 8C2) but were negative for leucocyte common antigen (CD45), Leu-6 (CD1), and the myelomonocytic L1 antigen. Characterization of HLA-DR positive DC isolated by an immunomagnetic bead method confirmed the immunohistochemical staining pattern that corresponds to the phenotype of interdigitating cells. Morphological and immunological implications of the abundant presence of these cells in pleomorphic adenomas are discussed.     

Fine needle aspiration of Langerhans cell histiocytosis of the lymph nodes. A report of six cases

BACKGROUND: Fine needle aspiration (FNA) diagnosis of Langerhans cell histiocytosis (LCH) of the lymph nodes has been described rarely. CASES: LCH was confined to the lymph nodes in six children. The FNA smears showed high cellularity composed of many isolated Langerhans cells (LCs) with nuclear grooves and intranuclear inclusions. Also seen were numerous eosinophils, lymphocytes, giant cells, dendriticlike cells, macrophages and Charcot-Leyden crystals in a background of eosinophilic granules. Immunohistochemical study revealed a positive reaction with S-100 protein. CONCLUSION: The presence of LCs with nuclear grooves, eosinophils, giant cells and a positive reaction with S-100 protein aided the diagnosis of LCH of the lymph nodes. Charcot-Leyden crystals, intranuclear inclusions and dendriticlike cells were seen in this study. These findings have not been reported before in lymph node FNA smears of LCH.     

IgE-mediated mast cell activation induces Langerhans cell migration in vivo

Langerhans cells and mast cells are both resident in large numbers in the skin and act as sentinel cells in host defense. The ability of mast cells to induce Langerhans cell migration from the skin to the draining lymph node in vivo was examined. Genetically mast cell-deficient (W/Wv) mice and control mice were sensitized with IgE Ab in the ear pinna. Seven to 14 days later, mice were challenged with Ag i.v. After a further 18-24 h, epidermal sheets and draining auricular lymph nodes were examined using Langerin/CD207 immunostaining. In mast cell-containing mice, a significant decrease in the number of Langerhans cells was observed at epidermal sites of mast cell activation. A significant increase in total cellularity and accumulation of Langerin-positive dendritic cells was observed in the auricular lymph nodes, draining the sites of IgE-mediated mast cell activation. These changes were not observed in W/Wv mice, but were restored by local mast cell reconstitution. Treatment of mast cell-containing mice with the H2 receptor antagonist cimetidine significantly inhibited the observed IgE/Ag-induced changes in Langerhans cell location. In contrast, Langerhans cell migration in response to LPS challenge was not mast cell dependent. These data directly demonstrate the ability of mast cells to induce dendritic cell migration to lymph nodes following IgE-mediated activation in vivo by a histamine-dependent mechanism.     

Lymph node resident rather than skin-derived dendritic cells initiate specific T cell responses after Leishmania major infection

Langerhans cells have been thought to play a major role as APCs for induction of specific immune responses to Leishmania major. Although their requirement for control of infection has been challenged recently, it remains unclear whether they can transport Ag to lymph nodes and promote initiation of T cell responses. Moreover, the role of dermal dendritic cells (DCs), another population of skin DCs, has so far not been addressed. We have investigated the origin and characterized the cell population responsible for initial activation of L. major-specific T cells in susceptible and resistant mice. We found that Ag presentation in draining lymph nodes peaks as early as 24 h after infection and is mainly mediated by a population of CD11c(high)CD11b(high)Gr-1-CD8-langerin- DCs residing in lymph nodes and acquiring soluble Ags possibly drained through the conduit network. In contrast, skin-derived DCs, including Langerhans cells and dermal DCs, migrated poorly to lymph nodes and played a minor role in early T cell activation. Furthermore, prevention of migration through early removal of the infection site did not affect Ag presentation by CD11c(high) CD11b(high) DCs and activation of Leishmania major-specific naive CD4+ T cells in vivo.     

前列腺癌微环境中树突状细胞与各类血细胞的关系及其临床预后价值的研究

探讨前列腺癌微环境中DCs与各类血细胞的关系及临床预后价值.选取16例良性前列腺增生和42例前列腺癌患者的前列腺组织作为研究对象,以S-100、CD83、CD208抗体作为不同状态的DC标记物进行MaxVision法免疫组化染色和Masson染色.采用图像分析软件进行图像处理,其统计数据与患者外周血细胞计数进行统计学分析.S-100、CD83阳性细胞计数和胶原蛋白含量在前列腺增生组较前列腺癌组高(P<0.05).CD208阳性细胞计数在前列腺增生组和前列腺癌组无差异(P>0.05).S-100阳性细胞计数与Gleason评分呈负相关关系(r=-0.533,P<0.01).血小板计数在前列腺癌组较前列腺增生组高(P<0.05).单核细胞计数偏高为前列腺癌危险因素(P<0.05).各类型树突状细胞与血小板计数无直线相关关系(P>0.05).外周血各成熟类型细胞与前列腺癌微环境中DCs计数无明显相关关系.S-100标记的树突状细胞计数可能与前列腺癌患者的预后相关.更大量样本的分析有助于证实单核细胞计数与前列腺癌的发病以及S-100标记树突状细胞计数与前列腺癌的预后之间的相关性.     

Characterization of dendritic cells,isolated from normal and stimulated lymph nodes of the rat

Summary Non-lymphoid dendritic cells were isolated from normal and paratyphoid vaccine-stimulated lymph nodes draining the rat skin. They were studied using enzymecytochemical, immunocytochemical and electron-microscopical methods. These cells had an irregular outline and an eccentrically situated nucleus. All showed acid phosphatase activity in a central area and expressed Ia antigen on the plasma membrane. Birbeck granules were exclusively present in dendritic cells isolated from lymph nodes in the induction phase of the immune response. This observation concurs with the presence of Birbeck granules in interdigitating cells in situ during the same period of the immune response. It is concluded that the dendritic cells are the in-vitro equivalents of the non-actively phagocytizing population of interdigitating cells.     

Immunohistochemical study on the distribution of alpha and beta subunits of S-100 protein in human neoplasm and normal tissues

The immunohistochemical distribution and localization of the alpha and beta subunits of S-100 protein in human neoplasms and normal tissues were studied by the PAP method using monospecific rabbit antibodies against each subunit. Beta subunit immunoreactivity was detected in all S-100-positive cells and tumors reported previously. In contrast alpha subunit immunoreactivity was absent from Schwann cells, schwannomas, neurofibromas, granular cell myoblastomas, pituicytes of the neurohypophysis, Langerhans cells, interdigitating reticulum cells, and histiocytosis X cells. Interestingly, only the alpha subunit was detected in neurons of both central and peripheral nervous system, and in lymph node macrophages. Human S-100-positive cells are divided into three groups; the first is composed of cells containing only the beta subunit (probably S-100b; beta beta), the second consists of cells containing both the alpha and beta subunits, and the third is composed of cells containing only the alpha subunit (probably S- 100ao ; alpha alpha). The ontogentic relationships between S-100-positive cells and tumors are discussed in the light of these findings.     

Targeting of epidermal Langerhans cells with antigenic proteins: attempts to harness their properties for immunotherapy

Langerhans cells, a subset of skin dendritic cells in the epidermis, survey peripheral tissue for invading pathogens. In recent functional studies it was proven that Langerhans cells can present exogenous antigen not merely on major histocompatibility complexes (MHC)-class II molecules to CD4⁺ T cells, but also on MHC-class I molecules to CD8⁺ T cells. Immune responses against topically applied antigen could be measured in skin-draining lymph nodes. Skin barrier disruption or co-application of adjuvants was required for maximal induction of T cell responses. Cytotoxic T cells induced by topically applied antigen inhibited tumor growth in vivo, thus underlining the potential of Langerhans cells for immunotherapy. Here we review recent work and report novel observations relating to the potential use of Langerhans cells for immunotherapy. We investigated the potential of epicutaneous immunization strategies in which resident skin dendritic cells are loaded with tumor antigen in situ. This contrasts with current clinical approaches, where dendritic cells generated from progenitors in blood are loaded with tumor antigen ex vivo before injection into cancer patients. In the current study, we applied either fluorescently labeled protein antigen or targeting antibodies against DEC-205/CD205 and langerin/CD207 topically onto barrier-disrupted skin and examined antigen capture and transport by Langerhans cells. Protein antigen could be detected in Langerhans cells in situ, and they were the main skin dendritic cell subset transporting antigen during emigration from skin explants. Potent in vivo proliferative responses of CD4⁺ and CD8⁺ T cells were measured after epicutaneous immunization with low amounts of protein antigen. Targeting antibodies were mainly transported by langerin⁺ migratory dendritic cells of which the majority represented migratory Langerhans cells and a smaller subset the new langerin⁺ dermal dendritic cell population located in the upper dermis. The preferential capture of topically applied antigen by Langerhans cells and their ability to induce potent CD4⁺ and CD8⁺ T cell responses emphasizes their potential for epicutaneous immunization strategies. This article is a symposium paper from the conference “Immunotherapy—From Basic Research to Clinical Applications,” Symposium of the Collaborative Research Center (SFB) 685, held in Tübingen, Germany, 6–7 March 2008.     

The Langerhans cell: from in vitro production to use in cellular immunotherapy

Dendritic cells constitute a family of antigen presenting cells defined by their morphology and their capacity to initiate primary immune response. Langerhans cells are paradigmatic dendritic cells, described in 1868 by a young medical student, Paul Langerhans in Berlin. Langerhans cells are present with epithelial cells in the epidermis, bronchi and mucosae. After antigenic challenge, Langerhans cells migrate into the T cell areas of proximal lymph nodes where they act as professional antigen-presenting cells. Langerhans cells originate in the bone marrow and CD34+ hematopo?etic progenitors are present in cord blood or circulating blood. They are actively involved in skin lesions of allergic contact dermatitis or atopic dermatitis, in cancer immunosurveillance and are infected by HIV in AIDS. Since 1992, Langerhans cells may be generated in vitro from CD34+ cord blood or circulating blood progenitors by culture with GM-CSF and TNF alpha, as well as from peripheral blood monocytes by culture with GM-CSF, IL4 and TGF beta 1. The possibility to obtain from the blood, the circulating progenitors of dendritic cells and the subsequent possibility to harvest a large number of these cells through in vitro culture using growth factors, have given rise to several very interesting therapeutic perspectives, especially in the field of anti-cancer immunotherapy. In dermatology advanced studies have concerned malignant melanomas. Anti-melanoma immunization trials were performed in patients, through dendritic cells charged with melanoma antigens. Side effects appear to be limited. Injections of antigenically charged dendritic cells were performed subcutaneously, intravenously or in the lymph nodes. Positive clinical responses were obtained with, in some cases, complete remission of the metastasis. These results open a particularly interesting perspective in the field of cancer treatment.     

Immunization with DNA through the skin

The skin has evolved as a barrier to prevent external agents, including pathogens, from entering the body. It has a complex and efficient immune surveillance system, which includes Langerhans cells and dendritic cells. By targeting the body's natural defense system, skin-DNA immunization attempts to produce an efficient immune response. Nucleic acid vaccines provide DNA for protein expression in a variety of cells, including keratinocytes, Langerhans cells, and dendritic cells, which are located in the two main areas of the skin, the epidermis (the most superficial layer) and the dermis. After maturation, Langerhans cells and dermal dendritic cells can migrate to local lymph nodes where presentation of antigens to T cells can occur and thus start a variety of immunologic responses. Dermal immunization methods described in this article target the epidermis, the dermis, or both and include: (a) stripping; (b) chemical modification; (c) trans-epidermal immunization (transcutaneous immunization or non-invasive vaccination of the skin); (d) gene gun technology; (e) electroporation; (f) intradermal injections; and (g) microseeding. These techniques all require the removal of hair, the circumvention or modification of the stratum corneum layer of the epidermis, and the addition of DNA or amplification of DNA signal. As the biology of the skin and the mechanisms of DNA vaccination are elucidated, these skin immunization techniques will be optimized. With refinement, skin-DNA immunization will achieve the goal of producing a reliable and efficacious immune response to a variety of pathogens.     

S-100-immunoreactive giant macrophages in lymphoid tissues of the guinea pig

Giant cells containing S-100 protein of the lymphoid tissues in the guinea pig were studied by immunohistochemistry using S-100 antiserum. S-100-immunoreactive giant cells were dendritic in shape, contained one or two irregular-shaped, euchromatic nuclei, phagosomes of various diameter, numerous mitochondria and microfilaments in the perikaryon, and extended cell processes free of cell organelles. These cells predominantly lined the superficial cortex facing the subcapsular sinus, were less numerously scattered in the medulla of lymph nodes and located at the marginal zone of the spleen. They also stained with S-100 alpha monoclonal antiserum and showed active phagocytosis for aldehyde-fixed red cells or colloidal carbon in the popliteal lymph node and spleen. S-100-immunoreactive giant cells also appeared in the corticomedullary zone of the thymus and in the interfollicular area of the Peyer's patches of the gut. Small sinus macrophages, which exhibited active phagocytosis for colloidal carbon but were less active for red cells in the popliteal lymph node and spleen, were not stained with S-100 antiserum. These findings indicate that S-100-immunoreactive giant cells of the lymph node and spleen are a subpopulation of macrophages different from S-100-negative cells of the small type.     

An Essential Role for CD44 Variant Isoforms in Epidermal Langerhans Cell and Blood Dendritic Cell Function

Upon antigen contact, epidermal Langerhans cells (LC) and dendritic cells (DC) leave peripheral organs and home to lymph nodes via the afferent lymphatic vessels and then assemble in the paracortical T cell zone and present antigen to T lymphocytes. Since splice variants of CD44 promote metastasis of certain tumors to lymph nodes, we explored the expression of CD44 proteins on migrating LC and DC. We show that upon antigen contact, LC and DC upregulate pan CD44 epitopes and epitopes encoded by variant exons v4, v5, v6, and v9. Antibodies against CD44 epitopes inhibit the emigration of LC from the epidermis, prevent binding of activated LC and DC to the T cell zones of lymph nodes, and severely inhibit their capacity to induce a delayed type hypersensitivity reaction to a skin hapten in vivo. Our results demonstrate that CD44 splice variant expression is obligatory for the migration and function of LC and DC.