Frequency Of Langerhans Cells Research Articles

350 Histone demethylase LSD1 is required for LC embryonic development but dispensable for LC maintenance and repopulation

Langerhans cells (LCs), the sole dendritic cell subpopulation in the epidermis, are potent regulators of immune surveillance and tolerance. Unlike conventional DCs, LCs follow unique patterns of development and maintenance under steady and inflamed states. Lysine-specific demethylase 1 (LSD1), known to mediate the demethylation of lysine amino acids on histone proteins, plays a key role in the maintenance and differentiation of hematopoietic stem cells. However, the role of LSD1 in LC ontogeny and homeostasis remains unknown. To address this, we generated Csf1rCreLSD1fl/fl conditional knockout (Csf1r.LSD1-KO) mice in which LSD1 was deficient in macrophage/monocytes and LC precursors from embryonic stage. We found a robust reduction of LC precursors in Csf1r.LSD1-KO mice at embryonic day 18.5 and postnatal day 0, suggesting that LSD1 is required for LC ontogeny at late embryonic stage. To investigate whether LSD1 is involved in LC maintenance and repopulation, we created CD11cCreLSD1fl/fl conditional knockout (CD11c.LSD1-KO) mice in which LSD1 was deficient in all DC populations including epidermal LCs after birth. Flow cytometry and immunofluorescent staining of skin showed that the frequency and number of LCs were unaltered in 2-week-old and adult CD11c.LSD1-KO mice compared to wild-type mice. Using an ultraviolet C (UVC)-induced skin damage model, we found that long-term LCs were able to repopulate to the inflamed epidermis of CD11c.LSD1-KO mice 2 weeks after UVC treatment. Overall, our data suggests that LSD1 is critical for LC embryonic development, but not required for the LC maintenance at steady state and repopulation under inflammatory conditions.

Langerhans cells in hypospadias: an analysis of Langerin (CD207) and HLA-DR on epidermal sheets and full thickness skin sections

BackgroundHypospadias are among the most common genital malformations. Langerhans Cells (LCs) play a pivotal role in HIV and HPV infection. The migration of LC precursors to skin coincides with the embryonic period of hypospadias development and genetic alterations leading to the formation of hypospadias impact the development of ectodermally derived tissues. We hypothesized that this might be associated with a difference in frequency or morphology of epidermal and dermal LCs in hypospadias patients.MethodsA total of 43 patients from two centers were prospectively included into this study after parental consent and ethics approval. Epidermal and dermal sheets were prepared from skin samples of 26 patients with hypospadias, 13 patients without penile malformations and 4 patients with penile malformations other than hypospadias. Immunofluorescence staining of sheets was performed with anti-HLA-DR-FITC and anti-CD207/Langerin-A594 antibodies. Skin sections from 11 patients without penile malformation and 11 patients with hypospadias were stained for Langerin. Frequencies as well as morphology and distribution of epidermal and dermal LCs on sheets and sections were microscopically evaluated. Cell counts were compared by unpaired t-tests.ResultsThere was no difference in frequency of epidermal LCs, Neither on sheets (873 ± 61 vs. 940 ± 84LCs/mm2, p = 0.522) nor on sections (32 ± 3 vs. 30 ± 2LCs/mm2, p = 0.697). Likewise, the frequency of dermal LCs (5,9 ± 0,9 vs. 7.5 ± 1.3LCs/mm2, p = 0.329) was comparable between patients with hypospadias and without penile malformation. No differences became apparent in subgroup analyses, comparing distal to proximal hypospadias (p = 0.949), younger and older boys (p = 0.818) or considering topical dihydrotestosterone treatment prior to surgery (p = 0.08). The morphology of the LCs was not different comparing hypospadias patients with boys without penile malformations.ConclusionsLCs are present in similar frequencies and with a comparable morphology and distribution in patients with hypospadias as compared to children without penile malformations. This suggests that patients with hypospadias are not different from patients with normal penile development considering this particular compartment of their skin immunity.

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.

Aging affects epidermal Langerhans cell development and function and alters their miRNA gene expression profile

Immunosenescence is a result of progressive decline in immune system function with advancing age. Epidermal Langerhans cells (LCs), belonging to the dendritic cell (DC) family, act as sentinels to play key roles in the skin immune responses. However, it has not been fully elucidated how aging affects development and function of LCs. Here, we systemically analyzed LC development and function during the aging process in C57BL/6J mice, and performed global microRNA (miRNA) gene expression profiles in aged and young LCs. We found that the frequency and maturation of epidermal LCs were significantly reduced in aged mice starting at 12 months of age, while the Langerin expression and ability to phagocytose Dextran in aged LCs were increased compared to LCs from < 6 month old mice. The migration of LCs to draining lymph nodes was comparable between aged and young mice. Functionally, aged LCs were impaired in their capacity to induce OVA-specific CD4+ and CD8+ T cell proliferation. Furthermore, the expression of miRNAs in aged epidermal LCs showed a distinct profile compared to young LCs. Most interestingly, aging-regulated miRNAs potentially target TGF-β-dependent and non- TGF-β-dependent signal pathways related to LCs. Overall, our data suggests that aging affects LCs development and function, and that age-regulated miRNAs may contribute to the LC developmental and functional changes in aging.

Impact of Varicella-Zoster Virus on Dendritic Cell Subsets in Human Skin during Natural Infection

Varicella-zoster virus (VZV) causes varicella and herpes zoster, diseases characterized by distinct cutaneous rashes. Dendritic cells (DC) are essential for inducing antiviral immune responses; however, the contribution of DC subsets to immune control during natural cutaneous VZV infection has not been investigated. Immunostaining showed that compared to normal skin, the proportion of cells expressing DC-SIGN (a dermal DC marker) or DC-LAMP and CD83 (mature DC markers) were not significantly altered in infected skin. In contrast, the frequency of Langerhans cells was significantly decreased in VZV-infected skin, whereas there was an influx of plasmacytoid DC, a potent secretor of type I interferon (IFN). Langerhans cells and plasmacytoid DC in infected skin were closely associated with VZV antigen-positive cells, and some Langerhans cells and plasmacytoid DC were VZV antigen positive. To extend these in vivo observations, both plasmacytoid DC (PDC) isolated from human blood and Langerhans cells derived from MUTZ-3 cells were shown to be permissive to VZV infection. In VZV-infected PDC cultures, significant induction of alpha IFN (IFN-alpha) did not occur, indicating the VZV inhibits the capacity of PDC to induce expression of this host defense cytokine. This study defines changes in the response of DC which occur during cutaneous VZV infection and implicates infection of DC subtypes in VZV pathogenesis.

Characterization of langerhans cells in epidermal sheets along the body of Armadillo ( Dasypus novemcinctus)

Armadillos are apparently important reservoirs of Mycobacterium leprae and an animal model for human leprosy, whose immune system has been poorly studied. We aimed at characterizing the armadillo's langerhans cells (LC) using epidermal sheets instead of tissue sections, since the latter restrict analysis only to cut-traversed cells. Epidermal sheets by providing an en face view, are particularly convenient to evaluate dendritic morphology (cells are complete), spatial distribution (regular vs. clustered), and frequency (cell number/tissue area). Lack of anti-armadillo antibodies was overcome using LC-restricted ATPase staining, allowing assessment of cell frequency, cell size, and dendrites extension. Average LC frequency in four animals was 528 LC/mm 2, showing a rather uniform non-clustered distribution, which increased towards the animal's head, while cell size increased towards the tail; without overt differences between sexes. The screening of antibodies to human DC (MHC-II, CD1a, langerin, CD86) in armadillo epidermal sheets, revealed positive cells with prominent dendritic morphology only with MHC-II and CD86. This allowed us to test DC mobilization from epidermis into dermis under topical oxazolone stimulation, a finding that was corroborated using whole skin conventional sections. We hope that the characterization of armadillo's LC will incite studies of leprosy and immunity in this animal model.

Measurement of cytokine expression and Langerhans cell migration in human skin following suction blister formation.

Contact allergen-induced migration of epidermal Langerhans cells (LCs) to draining lymph nodes is dependent upon receipt by LCs of at least two cytokine signals provided by tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta. It has been reported previously that intradermal injection of healthy human volunteers with homologous TNF-alpha or IL-1beta each induces a significant reduction in LC frequency, as measured in epidermal sheets prepared from 6-mm punch biopsies. In the current experiments, we have compared the frequency of LCs in punch biopsies with those obtained concurrently in epidermal sheets from the roofs of suction blisters isolated from the sun-protected buttock skin of healthy adult volunteers. There was a significant, approximately 30%, reduction in CD1a(+) LC numbers in suction blister roofs compared with punch biopsies. Injection of homologous recombinant IL-1beta, a stimulus that provokes measurable epidermal LC mobilization in punch biopsy sites, failed to provoke further LC migration in suction blister sites. These data suggest that the mechanical trauma to the skin caused by the creation of suction blisters provokes the degree of cutaneous inflammation necessary for LC mobilization. The responsive cells (only a proportion of resident LCs, approximately 30%) have already migrated, thus addition of an exogenous cytokine signal (IL-1beta) is without further effect. It is not possible therefore to measure the regulation of LC mobilization by exogenous cytokines in suction blister roofs. However, this technique provides an opportunity to profile induced changes in the cutaneous cytokine environment, with cytokine expression measured by a multiple cytokine array system. Using this technique, intradermal injection of IL-1beta was found to cause a marked upregulation of proinflammatory cytokines including TNF-alpha, IL-6, IL-8, monocyte chemotactic protein-1 (MCP-1) and the anti-inflammatory cytokine IL-10 in fluid from suction blisters raised at the site of injection. In conclusion, the suction blister technique appears to be a powerful tool for measurement of induced changes in cutaneous cytokines.

ICSBP is critically involved in the normal development and trafficking of Langerhans cells and dermal dendritic cells

AbstractInterferon consensus sequence-binding protein (ICSBP) is a transcription factor belonging to the interferon regulatory factor (IRF) family, recently shown to play a critical role in dendritic cell (DC) differentiation. Here, we analyzed the role of ICSBP in the development and trafficking of epidermal Langerhans cells (LCs) and dermal DCs and the implications for initiation of a competent immune response. ICSBP-/- mice exhibited a reduced frequency of LCs and a delayed mobility of DCs from skin that reflected a slower turnover rate in lymph nodes during steady-state conditions. Even under inflammatory changes, ICSBP-/- DCs displayed reduced mobility from skin to lymph nodes and, as a consequence, failed to induce a contact hypersensitivity (CHS) response, suggesting that these DCs were unable to initiate a competent antigen (Ag)–specific T-cell–mediated immunity. Moreover, bone marrow (BM)–derived DCs from ICSBP-/- mice exhibited an immature phenotype and a severe reduction of interleukin 12 (IL-12) expression. These BM DCs also showed a marked defect in their migratory response to macrophage inflammatory protein 3α (MIP-3α), MIP-3β, and the CC chemokine CCL21/6Ckine, which was paralleled by an impaired expression of the CC chemokine receptors, CCR6 and CCR7. Together, these results indicate that ICSBP is critically required for the development and trafficking of skin DCs, thus playing a critical role in the DC-mediated initiation of T-cell immunity.

Tumour necrosis factor-alpha-induced migration of human Langerhans cells: the influence of ageing.

Langerhans cells (LCs) play essential roles in the initiation and regulation of cutaneous immune responses mediated through their successful migration from the epidermis to draining lymph nodes while carrying antigen. Tumour necrosis factor (TNF)-alpha, a keratinocyte-derived cytokine, has recently been shown to play an important role in the mobilization of LCs from human epidermis. Although it is known that with age the immune system changes, the influence of increasing age on the function of human LCs has not been defined clearly. To examine the influence of age on the ability of TNF-alpha to induce LC migration. Ten elderly (six men, four women; mean age 76 years, range 72-79) and 10 young (six men, four women; mean age 23 years, range 18-35) volunteers received intradermal injections of 200 U of human recombinant TNF-alpha diluted in sterile saline, and control injections of sterile saline alone, at each of two paired sites identified on photoprotected buttock skin. Two hours later, paired injection sites were excised by punch biopsy. One set of paired biopsies was processed for assessment of the frequency and morphology of epidermal LCs, following preparation of epidermal sheets and immunofluorescence staining for the LC marker CD1a. The remaining paired biopsies were processed in formalin and the inflammatory response to TNF-alpha was assessed by standard histological examination. Mean +/- SEM baseline values for LC frequency within epidermal sheets were significantly different between young (1156.3 +/- 38.5 cells mm(-2)) and elderly subjects (835.7 +/- 48.2 cells mm(-2); P < 0.01). Intradermal injections of 200 U of TNF-alpha caused a significant reduction in the frequency of LCs in both elderly and young subjects (P < 0.01). However, the extent of TNF-alpha-induced LC migration was substantially different between the two groups, with a mean 9% reduction in LC frequency in elderly volunteers compared with a mean 23% decrease in young subjects. Exposure to TNF-alpha was associated with a perivascular polymorphonuclear infiltrate at 2 h in all young subjects; in contrast, only 50% of the elderly individuals showed evidence of such a response. There are significant differences between young and old skin with respect to both resting LC numbers and their response to TNF-alpha. These age-related changes in LC frequency and function may contribute to the altered cutaneous immune function observed in the elderly.

Induction by tumour necrosis factor alpha of dose-related changes in Langerhans cell frequency in mice.

It has been demonstrated previously that tumour necrosis factor alpha (TNF-alpha) provides an important signal for the migration of epidermal Langerhans cells (LC) from the skin. Intradermal administration to mice of homologous recombinant TNF-alpha induces both a rapid reduction in the frequency of LC local to the site of exposure and, somewhat later, an accumulation of dendritic cells in draining lymph nodes. It has been proposed recently, however, that the influence of TNF-alpha on LC function may be dose-dependent in nature with lower concentrations inducing migration, but higher concentrations immobilizing LC in the epidermis. To investigate this proposal we examined the kinetics and dose-response relationships of TNF-alpha-induced LC migration in mice. At all concentrations tested (50, 150 or 300 ng/ear), intradermal exposure to TNF-alpha caused within 30 min a significant reduction in the frequency of MHC class II (Ia)+ LC within epidermal sheets. With the lower concentrations of TNF-alpha this effect was still apparent when LC were enumerated in the epidermis up to 4 h following cytokine treatment. In contrast, however, exposure of mice to 300 ng of TNF-alpha was consistently associated with a considerably less marked, and statistically insignificant, reduction in LC frequency by 4 h. These data indicate that at all concentrations of the cytokine examined here, TNF-alpha was able to stimulate a rapid (within 30 min) reduction in epidermal LC numbers, but that the rapidity with which the epidermis was repopulated following the initiation of LC migration was influenced by the concentration of TNF-alpha administered. It is suggested that TNF-alpha may influence not only the tempo of LC migration, but also the kinetics of epidermal repopulation.

Tumour necrosis factor-alpha induces Langerhans cell migration in humans.

The role of tumour necrosis factor (TNF)-alpha in the mobilization and migration of human epidermal Langerhans cells (LC) has been investigated. Intradermal injection of normal human volunteers with homologous recombinant TNF-alpha was found to cause a dose-dependent reduction in the frequency of LC within epidermal sheets 2 h later. Equivalent results were obtained when epidermal LC were identified on the basis of either CD1a or HLA-DR expression. At the dose of TNF-alpha used routinely (500 U), treatment resulted in an average reduction in LC density of approximately 24%. Treatment with TNF-alpha was associated with a perivascular polymorphonuclear infiltration at 2 h, but the epidermis appeared normal with neither fibrinoid necrosis nor vasculitis, and LC morphology was not affected significantly. These results demonstrate that TNF-alpha provides an important signal for LC migration in humans and is likely therefore to play a crucial part in the induction of cutaneous immune responses.

Modulation of MHC class II+ Langerhans cell numbers in corticosteroid treated epidermis by GM-CSF in combination with TNF-alpha.

It is important to understand how dendritic cells (DC) are recruited, maintained and stimulated to migrate from tissues to lymph nodes. This is because DC are potent initiators of primary immune responses and candidates for vaccine development. Identification of factors which could lead to increased numbers of DC in tissues could affect immune responses by modulating their interaction with antigen which penetrates the tissue. To identify cytokines which could increase DC in tissues we tested the ability of GM-CSF, TNF-alpha and IL-6 to partially prevent steroid depletion of Langerhans cells (LC) from the epidermis. Cytokines diluted in serum-containing medium were compared with cytokines diluted in albumin-containing, serum-free medium in order to determine a minimum combination of cytokines required to increase LC and the effect of serum on the LC-increasing activity of cytokines. In the presence of serum, GM-CSF or TNF-alpha could increase LC frequency compared to the control; but in the absence of serum neither of these cytokines were effective unless they were combined with each other. In the presence of serum the combination of GM-CSF with TNF-alpha was ineffective. The data support the hypotheses that GM-CSF and TNF-alpha are both important in regulating LC numbers in the epidermis in vivo. Serum may modulate how each of these cytokines, separately or in combination, affect LC frequency in the epidermis - GM-CSF and TNF-alpha separately probably interact with other factors present in serum to increase LC frequency, whereas in combination it is possible that these separate effects are cancelled in the presence of serum. TNF-alpha and GM-CSF together, in the absence of serum, form one combination of a minimum number of cytokines which can regulate LC frequency in the epidermis; and IL-6 alone, or in combination with GM-CSF, does not increase LC frequency.

Combined immunophenotyping and FISH with sex chromosome-specific DNA probes for the detection of chimerism in epidermal Langerhans cells after sex-mismatched bone marrow transplantation.

Langerhans cells (LC) of the skin represent bone marrow-derived dendritic antigen-presenting cells and are therefore important in pathophysiological processes such as rejection, graft-versus-host disease, and graft-versus-leukemia-reaction after bone marrow transplantation (BMT). For understanding of these diseases, the evaluation of the chimeric status of LC following BMT is of great interest. To analyze the sex chromosome constitution of LC in the skin, we established a modified and refined technique of combined immunophenotyping and fluorescence in situ hybridization (FISH) and investigated frozen sections of skin biopsies from nine patients after allogeneic sex-mismatched BMT and of two healthy donors for control. LC were specifically labeled using a fluorescent CD1 a antibody and hybridized simultaneously with X and Y chromosome-specific DNA probes. The results of this practical application on nine leukemia patients show the appearance of donor-type LC and the persistence of host-type LC at various times (36 up to 1395 days) after sex-mismatched BMT. Complete chimerism of LC could not be detected in any case. The frequency of recipient-specific LC ranged from 7% to 92% and showed no correlation with time postgrafting. We conclude from our results of 1461 analyzed LC that combined immunophenotyping and interphase cytogenetic analysis by FISH is the method of choice for the assessment of chimerism in a particular cell type after sex-mismatched BMT. Its practical application on other tissues affected by BMT-related pathophysiological processes reveals further knowledge of the time-dependent course of chimeric patterns after BMT.

Modulation of Ia+ Langerhans cell numbers in vivo by cultured epidermis derived supernatants and by GM‐CSF

This paper demonstrates that epidermal cells in culture produce an activity which can increase the frequency of Ia+ epidermal Langerhans cells (LC). This was achieved by treating mice topically with a mixture containing supernatant derived from primary culture of murine epidermis (ES) and a synthetic corticosteroid, triamcinolone acetonide (TAC). The presence of the supernatant in the mixture partially protected the Ia+ LC from depletion by the steroid. The Ia+ LC frequency increasing activity was measured as the difference between the Ia+ LC frequency due to treatment with steroid mixed with supernatant and the Ia+ LC frequency due to treatment with steroid mixed with negative control medium. The mean frequency of Ia+ LC in epidermis treated with TAC mixed with ES was 606(SD 43) cells/mm2, as compared with 486 (SD 68) cells/mm2 in the epidermis treated with TAC mixed with control medium. The activity appeared to be caused by (a) proteinaceous factor(s). A fraction of ES which was retained above a > or = 10 KDa molecular weight cut-off membrane was capable of partially protecting Ia+ LC frequency from TAC depletion. Supernatants from cultured lymph nodes, dermis as well as the squamous cell carcinoma lines T7 and T79, but not the human osteosarcoma cell-line 143B, also contained similar activities. We demonstrate that GM-CSF also increased the number of Ia+ epidermal LC when applied topically to mouse skin in this system. Therefore, using this Ia+ LC frequency modulation system, we propose that GM-CSF is one example of a cytokine which may be involved in the regulation of Ia+ LC numbers in epidermis and that epidermal cells produce factors which can increase the number of Ia+ LC.

Human Mucosal Langerhans Cells: Postmortem Identification of Regional Variations in Oral Mucosa

Modified ATPase histochemistry was used to identify and count Langerhans cells (LC) in autopsy tissue from 8 oral mucosal sites, 8-20 h postmortem. The specificity of the ATPase method was confirmed on serial sections with indirect immunofluorescence using monoclonal antibodies OKT6 and antihuman Ia. Average LC counts on ATPase-stained epithelial sheets from each of the 8 sites ranged from 160-550 LC/mm2. Nonkeratinized mucosae of the soft palate, ventral tongue, lip, and floor of the mouth had the highest counts (mean +/- SD, 508 +/- 110 LC/mm2, n = 24), and keratinized mucosae of the hard palate and gingiva had the lowest counts (201 +/- 97 LC/mm2; n = 8). LC frequency was variable in 2 sites: In the dorsal tongue, LC occurred on only one side of filiform papillae and were absent from regularly recurring areas of interpapillary epithelium. In the cheek mucosa, LC clustered around connective tissue papillae and their numbers showed marked individual variation (130-650 LC/mm2). The number of LC in nonkeratinized oral mucosa is approximately the same as in skin, but keratinized oral mucosa has fewer LC. The frequency of oral mucosal LC varies inversely with the degree of keratinization. There are regions of the oral mucosa that have no LC.