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.

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.

Dynamic Changes in Resident and Infiltrating Epidermal Dendritic Cells in Active and Resolved Psoriasis.

Epidermal Langerhans cells (LCs) are spatially separated from dermal dendritic cells (DCs) in healthy human skin. In active psoriasis, maintained by local production of IL-23 and IL-17, inflammatory DCs infiltrate both skin compartments. Here we show that CCR2+ epidermal DCs (eDCs) were confined to lesional psoriasis and phenotypically distinct from dermal DCs. The eDCs exceeded the number of LCs and displayed high expression of genes involved in neutrophil recruitment and the activation of keratinocytes and T cells. Resident LCs responded to toll-like receptor 4 and toll-like receptor 7/8 activation with increased IL-23 production, whereas eDCs additionally produced IL-1ß together with IL-23 and tumor necrosis factor. Psoriasis typically recur in fixed skin lesions. eDCs were absent from resolved psoriasis. Instead, LCs from anti-tumor necrosis factor-treated lesions retained high IL23A expression and responded to toll-like receptor stimulation by producing IL-23. Our results reveal phenotypic and functional properties of eDCs and resident LCs in different clinical phases of psoriasis, and the capacity of these cells to amplify the epidermal microenvironment through the secretion of IL-17 polarizing cytokines.

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.

In Vivo Imaging of Cutaneous DCs in Mice.

Varieties of cells orchestrate immune responses. To capture such dynamic phenomena, intravital imaging is an important technique, and it may provide substantial information that is not available using conventional histological analyses. Multiphoton microscopy enables the direct, three-dimensional, and minimally invasive imaging of biological samples with high spatiotemporal resolution, and it has now become the leading method for in vivo imaging studies. Here we describe a basic method for in vivo imaging of dendritic cells (DCs) in the mouse ear skin using multiphoton microscopy.

Laser Scanning In Vivo Confocal Microscopy of Clear Grafts after Penetrating Keratoplasty.

PURPOSE: To evaluate the changes of keratocytes and dendritic cells in the central clear graft by laser scanning in vivo confocal microscopy after penetrating keratoplasty (PK). METHODS: Thirty adult subjects receiving PK at Shandong Eye Institute and with clear grafts and no sign of immune rejection after surgery were recruited into this study, and 10 healthy adults were controls. The keratocytes and dendritic cells in the central graft were evaluated by laser scanning confocal microscopy, as well as epithelium cells, keratocytes, corneal endothelium cells, and corneal nerves (especially subepithelial plexus nerves). RESULTS: Median density of subepithelial plexus nerves, keratocyte density in each layer of the stroma, and density of corneal endothelium cells were all lower in clear grafts than in controls. The dendritic cells of five (16.7%) patients were active in Bowman's membrane and stromal membrane of the graft after PK. CONCLUSIONS: Activated dendritic cells and Langerhans cells could be detected in some of the clear grafts, which indicated that the subclinical stress of immune reaction took part in the chronic injury of the clear graft after PK, even when there was no clinical rejection episode.

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.

Tumor necrosis factor-alpha and interleukin-17 differently affects Langerhans cell distribution and activation in an innovative three-dimensional model of normal human skin.

Among the several cytokines involved in the psoriasis pathogenesis, tumor necrosis factor (TNF)-alpha and interleukin (IL)-17 play a central role. Many biomolecular steps remain unknown due to difficulty to obtain psoriatic models. To investigate the effect of TNF-alpha and IL-17 on the ultrastructure, immunophenotype, and number of epidermal Langerhans cells (LCs), human skin explants (n=7) were cultured air-liquid interface in a Transwell system. Four different conditions were used: medium alone (control), medium added with 100 ng/ml TNF-alpha or 50 ng/ml IL-17 or a combination of both cytokines. Samples were harvested 24 and 48 h after cytokine addition and were frozen. Samples harvested at 24h were also processed for transmission electron microscopy (TEM). By immunofluorescence analysis with anti-human Langerin antibody (three experiments/sample) we calculated the percentage of LCs/mm(2) of living epidermis after 24 and 48 h of incubation (considering control as 100%). At 24h LC number was significantly higher in samples treated with both cytokines (216.71+15.10%; p<0.001) and in TNF-alpha (125.74+26.24%; p<0.05). No differences were observed in IL-17-treated samples (100.14+38.42%). After 48 h, the number of epidermal Langerin-positive cells in IL-17- and TNF-alpha treated samples slightly decreased (94.99+36.79% and 101.37+23% vs. their controls, respectively). With the combination of both cytokines epidermal LCs strongly decreased (120+13.36%). By TEM, upon TNF-alpha stimulus LCs appeared with few organelles, mostly mitochondria, lysosomes, and scattered peripherical BGs. Upon IL-17 stimulus, LCs showed a cytoplasm with many mitochondria and numerous BGs close to the perinuclear space and Golgi apparatus, but also at the periphery, at the beginning of the dendrites. The addition of both cytokines did not affect LC ultrastructure. Our study showed that IL-17 induced significant changes in LC ultrastructure, while the combination of both cytokines seems to have a strong chemo-attractant effect on epidermal LCs, supporting the relevance of investigating the interplay between LCs and pro-inflammatory cytokines in the ongoing of the disease.

Confocal microscopy of epithelial and langerhans cells of the cornea in patients using travoprost drops containing two different preservatives.

The recently developed confocal cornea microscopy offers the opportunity to examine pathologies of the cornea and to gain insight into the activity of innate immunity. We aimed to investigate the corneal epithelial and Langerhans cell (LC) densities along with dry eye parameters in primary open-angle glaucoma (POAG) subjects, treated with either of two commercially available travoprost 0.004 % topical medications containing different preservatives. (1: benzalkonium chloride 0.015 % (TravBAK) and 2: polyquaternium-1 (PQ) 0.001 % (TravPQ). Consecutive case series of nineteen POAG patients on TravBAK (mean age: 64.8 ± 13.6 years), nineteen POAG patients on TravPQ (mean age: 66.8 ± 11.3 years) and nineteen age-matched healthy control subjects (63.8 ± 8.2 years). Ocular surface disease index (OSDI), lid parallel conjunctival folds (LIPCOF), Schirmer test (ST) and tear break up time (TBUT) were assessed, and then corneal epithelial and LC densities were investigated with confocal microscopy. Tear production was significantly reduced in both glaucoma patient groups compared to healthy individuals (p < 0.05). TBUT was significantly reduced and epithelial cell densities were significantly greater in patients treated with TravBAK compared to healthy individuals (p < 0.05 for all). LC densities were greater in both glaucoma groups compared to control subjects (p < 0.05 for all). Travoprost therapy may compromise ocular surface. The limited alertness of the corneal immune system found in patients with TravPQ can be considered as indicators of a less disturbed ocular surface and better controlled corneal homeostasis.

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.

3D visualization of epidermal Langerhans cells.

Langerhans cells (LCs) are intraepidermal dendritic cells that extend their dendrites between keratinocytes to form a dense network that covers the entire body. The mammalian epidermis has two diffusion barriers, the stratum corneum and tight junctions (TJs). In their resting state, LCs sit underneath these two barriers. Once activated, LCs elongate their dendrites to dock with, or penetrate through, TJs, which allows them to survey the environment between the two barriers. Here, we describe a method to visualize this dynamic interaction of TJs and LCs in 3D by using mouse ear epidermal sheets.

Langerhans cells in the human tympanic membrane in health and disease: a morphometric analysis.

HYPOTHESIS: The normal tympanic membrane contains Langerhans dendritic cells, and they play a role in the pathogenesis of chronic suppurative otitis media. BACKGROUND: The presence of Langerhans dendritic cells in the normal tympanic membrane is disputed. However, they have been identified in tympanic membranes of patients with otitis media. A quantitative analysis of the distribution and morphology of these cells in the types of chronic suppurative otitis media has not been undertaken. METHODS: Samples of normal cadaveric tympanic membranes and those from patients with chronic suppurative otitis media of the tubotympanic and atticoantral varieties were stained with the immunohistochemical marker CD1a. The number of cells per unit length of basement membrane, diameters of cells, and number and length of dendritic processes were compared between the groups. RESULTS: CD1a-positive Langerhans dendritic cells were present in the normal tympanic membrane. The number of cells per unit length of basement membrane, diameters of cells, and the length of dendritic processes increased significantly in tubotympanic disease and in atticoantral disease, the difference being more pronounced in the latter form of otitis media. CONCLUSION: Langerhans cells are present in the normal tympanic membrane, and they probably play differing roles in the pathogenesis of tubotympanic and atticoantral forms of chronic suppurative otitis media.

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.

Visualizing the innate and adaptive immune responses underlying allograft rejection by two-photon microscopy.

Transplant rejection involves a coordinated attack of the innate and the adaptive immune systems of the host. To investigate this dynamic process and the contributions of both donor and host cells, we developed an ear skin graft model suitable for intravital imaging. We found that donor dermal dendritic cells (DCs) migrated rapidly from the graft and were replaced by host CD11b(+) mononuclear cells. The infiltrating host cells captured donor antigen, reached the draining lymph node and cross-primed graft-reactive CD8(+) T cells. Furthermore, we defined the mechanisms by which host T cells target graft cells. We found that primed T cells entered the graft from the surrounding tissue and localized selectively at the dermis-epidermis junction. Later, CD8(+) T cells disseminated throughout the graft and many became arrested. These results provide insights into the antigen presentation pathway and the stepwise progression of CD8(+) T cell activity, thereby offering a framework for evaluating how immunotherapy might abrogate the key steps in allograft rejection.

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.

Contacts between mast cells and dendritic cells in the human skin.

Langerhans cells are a dendritic cell type characteristic of the epidermis. Since mast cells secrete molecules potentially influent on dendritic cell differentiation, we have addressed the degree of proximity between these two cell types in biopsies of skin diseases characterized by massive influx of dendritic cell precursors. By fluorescence microscopy, avidin labeled mast cells were found in contact with CD1a+ dendritic cells. By electron microscopy, contacts between mast cells and blunty dendritic cells were found in areas corresponding to those where CD1a+ cells were localized by immunohistochemistry. We propose that mast cells can induce the differentiation of precursors into Langerhans cells through both the release of short range acting soluble factors and contact-mediating plasma membrane molecules.