Intradermal injection of an anti-Langerin-HIVGag fusion vaccine targets epidermal Langerhans cells in nonhuman primates and can be tracked in vivo.

The development of new immunization strategies requires a better understanding of early molecular and cellular events occurring at the site of injection. The skin is particularly rich in immune cells and represents an attractive site for vaccine administration. Here, we specifically targeted vaccine antigens to epidermal Langerhans cells (LCs) using a fusion protein composed of HIV antigens and a monoclonal antibody targeting Langerin. We developed a fluorescence imaging approach to visualize, in vivo, the vaccine-targeted cells. Studies were performed in nonhuman primates (NHPs) because of their relevance as a model to assess human vaccines. We directly demonstrated that in NHPs, intradermally injected anti-Langerin-HIVGag specifically targets epidermal LCs and induces rapid changes in the LC network, including LC activation and migration out of the epidermis. Vaccine targeting of LCs significantly improved anti-HIV immune response without requirement of an adjuvant. Although the co-injection of the TLR-7/8 synthetic ligand, R-848 (resiquimod), with the vaccine, did not enhance significantly the antibody response, it stimulated recruitment of HLA-DR+ inflammatory cells to the site of immunization. This study allowed us to characterize the dynamics of early local events following the injection of a vaccine-targeted epidermal LCs and R-848.

Identification of Langerhans-like cells in the immunocompetent tissues of channel catfish, Ictalurus punctatus.

Dendritic cells (DCs) are the most powerful antigen presenting cells (APCs) that have a critical role in bridging innate and adaptive immune responses in vertebrates. Dendritic cells have been characterized morphologically and functionally in the teleost fish models such as rainbow trout, salmonids, medaka, and zebrafish. The presence of DCs with remarkable similarities to human Langerhans cells (LCs) has been described in the spleen and anterior kidney of salmonids and rainbow trout. However, there is no evidence of the presence of DCs and their role in channel catfish immunity. In this study, we assessed DC-like cells in the immunocompetent tissues of channel catfish by immunohistochemistry (IHC), flow cytometry and transmission electron microscopy (TEM). We identified Langerin/CD207+ (L/CD207+) cells in the channel catfish anterior kidney, spleen and gill by IHC. Moreover, we described the cells that resembled mammal LC DCs containing Birbeck-like (BL) granules in channel catfish spleen, anterior and posterior kidneys and gill by TEM. Our data suggest that cells with DC-like morphology in the immune related organs of catfish may share morphological and functional properties with previously reported DCs in teleost fish and mammals. More detailed knowledge of the phenotype and the function of catfish DCs will not only help gain insight into the evolution of the vertebrate adaptive immune system but will also provide valuable information for development and optimization of immunotherapies and vaccination protocols for aquaculture use.

Alterations in the secretory pattern of dermal dendritic cells following melanin uptake.

Under a variety of circumstances, melanin occurs in the dermal compartment of the skin, being mostly observed in cells that have been termed melanophages, some of which have been identified as dermal dendritic cells. We analysed changes in the expression and secretion pattern of cytokines by dendritic cells after the uptake of melanin from various sources. Dendritic cells were derived from human primary blood monocytes or from the human monocytic cell line THP-1. Melanin uptake increased the secretion of the chemokines MIP-1ß (CCL4) and MCP-1 (CCL2). The higher MIP-1ß secretion was accompanied by higher MIP-1ß gene expression. Elevation of MIP-1ß secretion was dependent on the uptake of melanin but could not be induced by the phagocytosis of latex beads, indicating that the phagocytic process itself was not sufficient to increase the secretion of this cytokine. The data thus show that the uptake of melanin changes the cytokine expression and secretion pattern of dendritic-like cells.

Dicer-dependent microRNAs control maturation, function, and maintenance of Langerhans cells in vivo.

Dendritic cells (DCs) are central for the induction of T cell immunity and tolerance. Fundamental for DCs to control the immune system is their differentiation from precursors into various DC subsets with distinct functions and locations in lymphoid organs and tissues. In contrast to the differentiation of epidermal Langerhans cells (LCs) and their seeding into the epidermis, LC maturation, turnover, and MHC class II Ag presentation capacities are strictly dependent on the presence of Dicer, which generates mature microRNAs (miRNAs). Absence of miRNAs caused a strongly disturbed steady-state homeostasis of LCs by increasing their turnover and apoptosis rate, leading to progressive ablation of LCs with age. The failure to maintain LCs populating the epidermis was accompanied by a proapoptotic gene expression signature. Dicer-deficient LCs showed largely increased cell sizes and reduced expression levels of the C-type lectin receptor Langerin, resulting in the lack of Birbeck granules. In addition, LCs failed to properly upregulate MHC class II, CD40, and CD86 surface molecules upon stimulation, which are critical hallmarks of functional DC maturation. This resulted in inefficient induction of CD4 T cell proliferation, whereas Dicer-deficient LCs could properly stimulate CD8 T cells. Taken together, Dicer-dependent generation of miRNAs affects homeostasis and function of epidermal LCs.

S100-negative indeterminate cell histiocytosis in an African American child responsive to narrowband ultraviolet B.

Indeterminate cell histiocytosis is a rare cutaneous disease characterized by the presence of dendritic cells that lack Birbeck granules and immunophenotypically shares features of both Langerhans cells and macrophages. We describe a case of a 4-year-old African American boy affected by a disseminated, exclusively mucocutaneous form of indeterminate cell histiocytosis. The eruption was successfully treated with narrowband ultraviolet B. The peculiar negativity of the Langerhans cell marker S100 is also discussed.

A case of cutaneous mastocytosis in a child with prominent Langerhans cell infiltration.

We recently encountered a 2-year-old boy with slightly infiltrative brown papules on the face, trunk, and extremities. Stroking of one of the papules produced an urticarial wheal (positive Darier's sign). Histopathologic tests revealed a dense infiltration of mast cells containing numerous granules and showing metachromasia under Toluidine blue staining. Immunohistochemical tests revealed that these cells were positive for CD68 and for c-kit. In addition, dermal dendritic cells that were positive for S100 and CD1a immunostaining were intermingled with the mast cells. We confirmed through electron microscopy that the dermal dendritic cells that were observed adjacent to the infiltration of mast cells had Birbeck granules in their cytoplasm, namely Langerhans cells. However, because of the greater numbers of mast cells than Langerhans cells, and because of the absence of both monomorphic LC proliferation and systemic symptoms of Langerhans cell histiocytosis, the present case favors a diagnosis of cutaneous mastocytosis in a child with Langerhans cell infiltration.

Phagocytosis of antigens by Langerhans cells in vitro.

Dendritic cells (DC) isolated from lymphoid tissues are generally thought to be nonphagocytic in culture. It has therefore been unclear how these cells could acquire particulate antigens such as microorganisms for initiation of primary immune responses. Lymphoid DC derive in part from cells that have migrated from nonlymphoid tissues, such as Langerhans cells (LC) of skin. The ability of LC to internalize a variety of particles was studied by electron, ultraviolet, phase, and differential interference contrast microscopy, and by two-color flow cytometry. Freshly isolated LC in epidermal cell suspensions phagocytosed the yeast cell wall derivative zymosan, intact Saccharomyces cerevisiae, representatives of two genera of Gram-positive bacteria, Corynebacterium parvum and Staphylococcus aureus, as well as 0.5-3.5-microns latex microspheres. During maturation in culture, the phagocytic activity of these cells was markedly reduced. Likewise, freshly isolated splenic DC were more phagocytic than cultured DC for two types of particle examined, zymosan and latex beads. Unlike macrophages, LC did not bind or internalize sheep erythrocytes before or after opsonization with immunoglobulin G or complement, and did not internalize colloidal carbon. The receptors mediating zymosan uptake by LC were examined. For this particle, C57BL/6 LC were considerably more phagocytic than BALB/c LC and exhibited a reproducible increase in phagocytic activity after 6 h of culture followed by a decline, whereas this initial rise did not occur for BALB/c LC. These differential kinetics of uptake were reflected in the pattern of zymosan binding at 4 degrees C, and endocytosis of the soluble tracer fluorescein isothiocyanate-mannose-bovine serum albumin at 37 degrees C. Zymosan uptake by LC from both strains of mice was inhibited in the presence of mannan or beta-glucan, although to different extents, but not by antibodies specific for CR3 (CD11b/CD18). These data indicate that zymosan uptake by LC can be mediated by a mannose/beta-glucan receptor(s) that is differentially expressed in the two strains of mice and that is downregulated during maturation of LC in culture.

Epidermal Langerhans cells from normal human skin bind monomeric IgE via Fc epsilon RI.

Human epidermal Langerhans cells (LC) bearing IgE are found in disease states associated with hyperimmunoglobulinemia E. When studying the mechanism(s) underlying this phenomenon, immunohistology revealed that a majority of epidermal LC from normal skin of healthy individuals can specifically bind monomeric IgE. IgE binding to LC could neither be prevented by preincubation of the tissue with monoclonal antibodies (mAb) against either Fc epsilon RII/CD23 or Fc gamma RII/CD32, nor by the addition of lactose. However, binding could be entirely abrogated by preincubation with the anti-Fc epsilon RI alpha mAb 15-1, which interferes with IgE binding to Fc epsilon RI alpha gamma transfectants. These observations indicated that IgE binding to epidermal LC is mediated by Fc epsilon RI rather than by CD23, CD32, or the D-galactose-specific IgE-binding protein. This assumption gained support from our additional findings that: (a) the majority of LC exhibited distinct surface immunolabeling with the anti-Fc epsilon RI alpha mAbs 15-1 and 19-1, but not with any of eight different anti-Fc epsilon RII/CD23 mAbs; and (b) transcripts for the alpha, beta, and gamma chains of Fc epsilon RI could be amplified by polymerase chain reaction from RNA preparations of LC-enriched, but not of LC-depleted, epidermal cell suspensions. In view of the preeminent role of Fc epsilon RI crosslinking on mast cells and basophils in triggering the synthesis and release of mediators of allergic reactions, the demonstration of this receptor on epidermal LC may have important implications for our understanding of allergic reactions after epicutaneous contact with allergens.

Intraepithelial airway dendritic cells: a distinct subset of pulmonary dendritic cells obtained by microdissection.

Dendritic cells (DC), in general, and pulmonary DC, in particular, are a heterogeneous population of cells, their phenotype and function being dependent on their anatomic location, their state of activation, and the regulatory effect of locally secreted cytokines. Using a novel microdissection technique, the epithelium from the trachea and entire airway system was harvested, and the contained DC isolated at greater than 90% purity. The phenotype and function of these airway DC (ADC) was compared to DC isolated, at greater than 90% purity, from the parenchyma of the same lung. In contrast to lung DC (LDC), ADC did not express intercellular adhesion molecule 1 (ICAM-1) in situ, the amount of immune associated antigen (Ia) expressed was less (as determined by immunoperoxidase staining and immunopanning), and greater than 50% of ADC displayed Fc receptors (FcR). The majority of LDC were ICAM-1+, less than 5% expressed FcR, and all were intensely Ia+. Airway DC were most numerous in tracheal epithelium, but they were also present in small numbers in the epithelium of the most distal airways. Their numbers increased in all segments of the tracheobronchial epithelium in response to the administration of IFN-gamma. ADC were consistently more effective than LDC in presenting soluble (hen egg lysozyme) and particulate (heat-killed Listeria monocytogenes) antigens to antigen-sensitized T cells. By contrast, LDC were significantly more efficient in stimulating the proliferation of nonsensitized T cells in an autologous mixed leukocyte reaction. These data suggest that in normal animals, intraepithelial DC of airways share many attributes with Langerhans cells of the skin. Interstitial LDC, by contrast, reside in an environment where they may be exposed to a different set of regulatory factors and where they have progressed to a more advanced stage of differentiation than ADC. Both groups of DC are, however, heterogeneous, reflecting the continuous turnover that these cells undergo in the lung.

Effect of glucocorticosteroids on epidermal Langerhans cells.

The effects of topical and systemic administration of various glucocorticoids on the density of epidermal Langerhans cells (LC) were studied in guinea pigs. Glucocorticoids, such as betamethasone dipropionate and valerate, caused a marked decrease in LC demonstrable by staining for cell membrane ATPase activity and Ia antigens. By electronmicroscopy, LC also showed morphologic alterations. The observed decrements in LC density correlated with the concentration and known vasoconstrictive potency of the glucocorticoids administered. The anti-inflammatory action of glucocorticoids in skin disorders may, at least in part, be through their ability to alter epidermal LC, thus interfering with the antigen-presenting functions of these cells.

Disappearance of certain acidic organelles (endosomes and Langerhans cell granules) accompanies loss of antigen processing capacity upon culture of epidermal Langerhans cells.

Freshly isolated epidermal Langerhans cells (LC) can actively process native protein antigens, but are weak in sensitizing helper T cells. During culture, when LC mature into potent immunostimulatory dendritic cells, T cell sensitizing capacity develops but antigen processing capacity is downregulated. Processing of exogenous antigens for class II-restricted antigen presentation involves acidic organelles. We used the DAMP-technique to monitor acidic organelles at the ultrastructural level in fresh, as well as cultured, mouse and human LC. We observed that the loss of antigen processing capacity with culture of LC was reflected by the disappearance of certain acidic organelles, namely endosomes (particularly early ones), and the hitherto enigmatic LC granules ("Birbeck Granules"). Our findings support the notion that endosomes are critical for antigen processing and suggest that LC granules might be involved as well.

Murine epidermal Langerhans cells mature into potent immunostimulatory dendritic cells in vitro.

Murine epidermal Langerhans cells (LC) have been studied in tissue culture and compared to spleen dendritic cells (DC). LC comprised 3% of the starting cell suspensions and were distinguished from keratinocytes by cytology and reactivity with anti-Ia and anti-Mac-1 monoclonal antibodies. The LC were nonadherent, had a low buoyant density, did not proliferate, and could be enriched to 10-50% purity. LC continued to exhibit Ia and Mac-1 antigens for 4 d in culture. However, LC rapidly lost Birbeck granules, Fc receptors, F4/80 antigen, and cytochemical reactivity for nonspecific esterase and membrane ATPase. As a result, the ultrastructure and phenotype of cultured LC became remarkably similar to lymphoid DC. Stimulatory capacity for T cell proliferative responses (oxidative mitogenesis and the mixed leukocyte reaction) was monitored daily. Initially, stimulatory capacity was very weak, even though LC expressed substantial levels of Ia antigens. After 2-3 d in culture, LC had become 3-10 times more potent than spleen DC. 30 LC could induce significant responses in cultures of 3 X 10(5) responding T cells. Removal of Ia+ LC at the start of culture ablated the development of stimulatory activity, but exposure to 1,500 rad of ionizing irradiation did not. Mixing experiments showed that contaminating Ia- epidermal cells did not alter the function of Ia+ stimulators. Therefore, LC seem to be immunologically immature, but acquire many of the features of spleen DC during culture. We suggest that functioning lymphoid DC may, in general, be derived from less mature precursors located in nonlymphoid tissues.

Size-dependent cytotoxic effects of amorphous silica nanoparticles on Langerhans cells.

Amorphous silica nanoparticles (nSPs), are widely used in medicines, cosmetics and food. However, due to their reduced particle size they are suspected to pose new risks induced by changes in biological reactivity and kinetics, which differ from those of bulk materials. In a previous study, we showed that silica particles with a diameter of 70 nm penetrated the stratum corneum (SC) of mouse skin and were taken up by living cells such as keratinocytes and Langerhans cells. To clarify the relationship between particle size, distribution and cellular response, we have evaluated size-dependent intracellular localization and cytotoxicity of silica particles, using the mouse epidermal Langerhans cell line XS52. On treatment with silica particles of diameters 70, 300, and 1000 nm, cellular uptake and cytotoxicity increased with reduction in particle size. These results suggest that smaller sized silica particles induced greater cytotoxicity against Langerhans cells, which was correlated with the quantity of particle uptake into the cells.

Reactivity of Langerhans cells after application of different chemicals--an ultrastructural analysis.

The goal of this study was to investigate changes in epidermal Langerhans cells after application of different chemicals (acetone, 60% alcohol, 5% nickel sulphate, iodisole, and 0.1% 2,4-dinitrochlorobenzene) on the skin of volunteers. The skin of eight volunteers was treated with acetone, 60% alcohol, iodisol, 5% nickel sulphate, and 0.1% 2,4-dinitrochlorobenzene (DNCB). After application of DNCB, Langerhans cells (LCs) showed increased accumulation of Birbeck granules (Bgs). Alcohol and nickel sulphate caused alternative changes, mainly cytoplasmic vacuolation, in LCs. Nickel sulphate was even responsible for the disappearance of dendrites. Both chemicals have cytotoxic effects on LCs: cytoplasmic organelles and Bgs disappear and subsequently, the antigen-presenting activity of epidermal LCs is inhibited. We did not found any morphological changes in LCs after application of acetone.

The Essential Role of anxA2 in Langerhans Cell Birbeck Granules Formation.

Langerhans cells (LC) are the resident antigen presenting cells of the mucosal epithelium and play an essential role in initiating immune responses. LC are the only cells in the body to contain Birbeck granules (BG), which are unique cytoplasmic organelles comprised of c-type lectin langerin. Studies of BG have historically focused on morphological characterizations, but BG have also been implicated in viral antigen processing which suggests that they can serve a function in antiviral immunity. This study focused on investigating proteins that could be involved in BG formation to further characterize their structure using transmission electron microscopy (TEM). Here, we report a critical role for the protein annexin A2 (anxA2) in the proper formation of BG structures. When anxA2 expression is downregulated, langerin expression decreases, cytoplasmic BG are nearly ablated, and the presence of malformed BG-like structures increases. Furthermore, in the absence of anxA2, we found langerin was no longer localized to BG or BG-like structures. Taken together, these results indicate an essential role for anxA2 in facilitating the proper formation of BG.

Uncommon cutaneous non-Langerhans cell histiocytosis arising from dermal dendritic cells in adults: a clinicopathological study of five cases.

BACKGROUND: Non-Langerhans cell histiocytosis (non-LCH) is a collective term that encompasses a long list of rare "histiocytosis" that do not meet the criteria for Langerhans cell histiocytosis (LCH). Among cutaneous non-LCH, the xanthogranuloma (XG) family represents a distinct group of disorders derived from dermal dendritic cells (DDCs) at different stages of differentiation. OBJECTIVES: To investigate the clinicopathological characteristics of the XG family in adults and review the relevant literature. MATERIALS AND METHODS: We performed a retrospective clinicopathological study of five adult cases with a previous diagnosis of non-LCH. Clinicopathological features, immunophenotypes, genetic alterations and ultrastructural characteristics were analysed. RESULTS: Skin biopsies revealed that all five cases were characterized by diffuse infiltration of polymorphic cells, which were immunoreactive to factor XIIIa but negative for Langerin, CD1a, and S100. None of the cases harboured the BRAF V600E mutation. Electron microscopy of two cases exhibited abundant cytoplasmic processes with numerous lysosome-like dense bodies and electron-lucent vesicles in the cytoplasm and extracellular matrix. The overall features suggested that DDCs are the cellular origin, and these cases fulfilled the criteria for the XG family. CONCLUSION: The XG family represents a spectrum of rare diseases with different clinical presentations, a wide range of morphological appearances, and a shared common origin (DDCs). This group of disorders has been proposed as a unique entity with diagnostic challenges that should not be underestimated.

Structural studies of langerin and Birbeck granule: a macromolecular organization model.

Dendritic cells, a sentinel immunity cell lineage, include different cell subsets that express various C-type lectins. For example, epidermal Langerhans cells express langerin, and some dermal dendritic cells express DC-SIGN. Langerin is a crucial component of Birbeck granules, the Langerhans cell hallmark organelle, and may have a preventive role toward HIV, by its internalization into Birbeck granules. Since langerin carbohydrate recognition domain (CRD) is crucial for HIV interaction and Birbeck granule formation, we produced the CRD of human langerin and solved its structure at 1.5 A resolution. On this basis gp120 high-mannose oligosaccharide binding has been evaluated by molecular modeling. Hydrodynamic studies reveal a very elongated shape of recombinant langerin extracellular domain (ECD). A molecular model of the langerin ECD, integrating the CRD structure, has been generated and validated by comparison with hydrodynamic parameters. In parallel, Langerhans cells were isolated from human skin. From their analysis by electron microscopy and the langerin ECD model, an ultrastructural organization is proposed for Birbeck granules. To delineate the role of the different langerin domains in Birbeck granule formation, we generated truncated and mutated langerin constructs. After transfection into a fibroblastic cell line, we highlighted, in accordance with our model, the role of the CRD in the membrane zipping occurring in BG formation as well as some contribution of the cytoplasmic domain. Finally, we have shown that langerin ECD triggering with a specific mAb promotes global rearrangements of LC morphology. Our results open the way to the definition of a new membrane deformation mechanism.

In vitro complement binding on cytoplasmic structures in normal human skin: immunoelectronmicroscopic studies.

We have previously provided evidence that suggests that exposure of cryostat skin sections to normal human serum (NHS) results in the antibody-independent Clq binding to cytoplasmic structures of various cell types, leading to classical complement pathway activation as evidenced by cytoplasmic C3 deposition. In the present study, we have employed immunoelectronmicroscopic methods to clarify the exact nature of cytoplasmic C3 binding structures. Incubation of cryostat skin sections with NHS followed by peroxidase-labeled rabbit anti-human C3 serum (HRP-R/Hu C3) revealed that intracytoplasmic binding of C3 occurred in suprabasal keratinocytes, melanocytes, fibroblasts, smooth muscle cells, endothelial cells, pericytes, Schwann cells, and nerve axons, but not in basal keratinocytes, Langerhans cells, and other cellular constituents of the skin. C3 binding, as revealed by the deposition of HRP reaction product, was exclusively confined to intermediate-sized filaments (ISF), which can therefore be considered to represent the subcellular site for classical complement pathway activation. Under experimental conditions that do not allow classical complement pathway activation, ISF were not decorated. Our observation that ISF of ontogenetically different cell types share the capacity of complement fixation is in accordance with the recent finding that different ISF types, despite their biochemical and antigenic heterogeneity, have common alpha-helical domains and may provide a clue to the mechanism and site of interaction between complement components and ISF.

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.

Autophagy links antimicrobial activity with antigen presentation in Langerhans cells.

DC, through the uptake, processing, and presentation of antigen, are responsible for activation of T cell responses to defend the host against infection, yet it is not known if they can directly kill invading bacteria. Here, we studied in human leprosy, how Langerhans cells (LC), specialized DC, contribute to host defense against bacterial infection. IFN-γ treatment of LC isolated from human epidermis and infected with Mycobacterium leprae (M. leprae) activated an antimicrobial activity, which was dependent on the upregulation of the antimicrobial peptide cathelicidin and induction of autophagy. IFN-γ induction of autophagy promoted fusion of phagosomes containing M. leprae with lysosomes and the delivery of cathelicidin to the intracellular compartment containing the pathogen. Autophagy enhanced the ability of M. leprae-infected LC to present antigen to CD1a-restricted T cells. The frequency of IFN-γ labeling and LC containing both cathelicidin and autophagic vesicles was greater in the self-healing lesions vs. progressive lesions, thus correlating with the effectiveness of host defense against the pathogen. These data indicate that autophagy links the ability of DC to kill and degrade an invading pathogen, ensuring cell survival from the infection while facilitating presentation of microbial antigens to resident T cells.