Langerhans Cells In Epidermal Sheets Research Articles

Differential immunological effects of infrared irradiation and its associated heat in vivo

Infrared irradiation (IR) is the most abundant fraction of sunlight reaching the earth's surface and provides heat. The fever response of an animal is known to regulate its immune responses. However, the non-thermal immune responses of IR were difficult to assess owing to its close association with heat. We hypothesized that IR irradiation induced differential immunological responses, independent of its associated heat. With an IR machine coupled with a delicate temperature control system, we investigated the non-thermal immunological effects of IR in vivo. With heating at 37 °C or 39 °C using an electric blanket or IR irradiation, we measured the skin's physiological parameters, including transepidermal water loss (TEWL), pH, skin hydration, elasticity, sebum production, and skin blood flow. We also measured the number of Langerhans cells in epidermal sheets and draining lymph nodes. Lymph node cells were activated by anti-CD3 antibody and their production of interleukin (IL)-5, 10, 13, 17, and interferon (IFN)-γ was measured by enzyme-linked immunosorbent assay (ELISA). The result showed that compared to heating alone, IR causes an enhanced activation of epidermal Langerhans cells, both in epidermal sheets and in draining lymph nodes. The activation of draining lymph node cells by anti-CD3 antibody in vitro induces both Th2 and Th1, but not Treg immune responses. Interestingly, IL-13, a Th2 cytokine, is induced the most. In contrast, physiological parameters and barrier functions of skin were not altered after IR irradiation. The study showed that IR alone without heat modulates immune responses in vivo, indicating that IR irradiation might regulate host immunity in a heat-independent manner.

Differential immunological effects of infrared irradiation and its associated heat in vivo

Infrared irradiation (IR) is the most abundant fraction of sunlight reaching the earth's surface and provides heat. The fever response of an animal is known to regulate its immune responses. However, the non-thermal immune responses of IR were difficult to assess owing to its close association with heat. We hypothesized that IR irradiation induced differential immunological responses, independent of its associated heat. With an IR machine coupled with a delicate temperature control system, we investigated the non-thermal immunological effects of IR in vivo. With heating at 37 °C or 39 °C using an electric blanket or IR irradiation, we measured the skin's physiological parameters, including transepidermal water loss (TEWL), pH, skin hydration, elasticity, sebum production, and skin blood flow. We also measured the number of Langerhans cells in epidermal sheets and draining lymph nodes. Lymph node cells were activated by anti-CD3 antibody and their production of interleukin (IL)-5, 10, 13, 17, and interferon (IFN)-γ was measured by enzyme-linked immunosorbent assay (ELISA). The result showed that compared to heating alone, IR causes an enhanced activation of epidermal Langerhans cells, both in epidermal sheets and in draining lymph nodes. The activation of draining lymph node cells by anti-CD3 antibody in vitro induces both Th2 and Th1, but not Treg immune responses. Interestingly, IL-13, a Th2 cytokine, is induced the most. In contrast, physiological parameters and barrier functions of skin were not altered after IR irradiation. The study showed that IR alone without heat modulates immune responses in vivo, indicating that IR irradiation might regulate host immunity in a heat-independent manner.

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.

Ontogeny of Langerin/CD207 Expression in the Epidermis of Mice

C-type lectin receptors help Langerhans cells (LC) to take up and process pathogens. Langerin/CD207 is a mannose-binding C-type lectin that is specifically expressed by LC. It is involved in antigen uptake in an as yet poorly defined way, and it is a major molecular constituent of Birbeck granules. We studied the emergence of Langerin expression in LC in epidermal sheets and cell suspensions during ontogeny. Langerin appears later than MHC II expression. Intracellular Langerin expression becomes apparent 2-3 d after birth. Only 10 days after birth all LC co-express Langerin. The intensity of Langerin expression reaches adult levels by 3 wk after birth. Early Langerin expression appears to correlate at least in part with the physical presence of Birbeck granules.

Exposure to multiple doses of UVB radiation reduces the numbers of epidermal Langerhans cells and lymph node dendritic cells in mice.

Immune suppression following UVB irradiation is partly attributed to the effects of the exposure on antigen-presenting cells. Following a single UVB irradiation, there is a decrease in epidermal Langerhans cell numbers; this is accompanied by an increase in the number of dendritic cells (DC) in lymph nodes draining the irradiated site. We investigated whether a similar effect occurred following multiple UVB exposures. Mice were irradiated on their ears and shaved dorsal skin twice a week for 3 weeks. After the final exposure, the number of ATPase(+) Langerhans cells in epidermal sheets prepared from the ears was found to be decreased by 33% compared to unirradiated controls. The number of DC in the draining lymph nodes (DLN) did not increase as might have been expected; rather, a significant decrease of approximately 30% in DC numbers in the DLN of UVB-irradiated mice compared with unirradiated controls occurred. This decrease in antigen-presenting cells in both the epidermis and the DLN may be an important contributing factor to the immune suppression that follows multiple UVB exposures.

Prolongation of skin allograft survival is associated with reduced Th1 cytokine responses in the WKY-->F344 rat model.

We have reported previously that F344 rats develop a spontaneous tolerance to WKY lung allografts and show long-term retention of donor-specific skin grafts placed 35 days after lung transplantation. In this study, we investigated the immunologic mechanisms that may be responsible for the prolonged skin graft survival in animals tolerized with lung allografts. In the rejection group, WKY skin grafts were placed on normal F344 rats, whereas, in the tolerance group, the skin grafts were placed on F344 rats that had received a WKY lung transplant 35 days before skin grafting. Th1 (interleukin [IL]-2 and interferon-gamma [IFN-gamma]) and Th2 (IL-4 and IL-10) cytokine as well as transforming growth factor-beta1 mRNA expression in skin grafts and in draining lymph nodes were determined by reverse transcription-polymerase chain reaction. Macrophage and lymphocyte infiltration in skin grafts and the number of Langerhans cells in epidermal sheets of the grafts were examined by immunohistochemistry. IL-2 and IFN-gamma mRNA expression was significantly decreased in both the skin grafts and the draining lymph nodes of the tolerance group, compared to the rejection group, whereas IL-10 and transforming growth factor-beta1 mRNA expression was similar in both groups and IL-4 mRNA was rarely detected. Decreased and delayed CD8+, macrophage, and natural killer cell infiltration in the skin grafts from the tolerance group was also detected. Similar reduction in the number of Langerhans cells in the epidermis of the grafts from both groups was seen on day 1 after skin grafting, and thereafter the number remained stable in both groups. Reduced expression of Th1 cytokines and decreased infiltration of CD8+ cells, macrophages, and natural killer cells in the skin grafts may be responsible for prolongation of skin graft survival in the tolerance group.

Heterogeneous reactivity of murine epidermal Langerhans cells after application of FITC: a histochemical evaluation.

To elucidate the detailed kinetics of epidermal Langerhans cells after topical contact sensitizer stimulation, we examined ATPase or Ia positive epidermal cells of BALB/c mice in a time-spaced manner after the topical application of fluorescein isothiocyanate (FITC). We also performed double labeling of Langerhans cells in epidermal sheets with ATPase activity and Ia antigen or costimulatory molecules (B7-1 and B7-2) after the same stimulation. Observations showed that the density of ATPase positive cells and Ia positive cells decreased following a different time course; the former reached a nadir (77.4% of control) at 4 h but the latter reached a minimum (82.8% of control) at 16 h after the application of FITC. A double labeling technique revealed an increase in Ia single positive cells at 4 h as opposed to that of ATPase single positive cells at 16 h after application. Both costimulatory molecules were expressed on the dendritic processes of many Langerhans cells as a dotty pattern at 4 h after application; B7 positive and ATPase negative areas were observed at this time. On electron microscopic observation, a few activated Langerhans cells found in the dermis at 4 h after application had distinctive profiles compared with residual Langerhans cells in the epidermis. These findings suggest that there is a heterogeneity of reactivity to FITC in epidermal Langerhans cells, and that only a small portion of them migrates from the epidermis during sensitization. The findings also indicate the importance of the interaction between the Langerhans cell and its surrounding microenvironment in the epidermis for its activation. In addition, the results indicate that the enzymatic and the phenotypic markers do not definitively reflect the presence (or absence) of Langerhans cells.

Entry Into Afferent Lymphatics and Maturation In Situ of Migrating Murine Cutaneous Dendritic Cells

An important property of dendritic cells (DC), which contributes crucially to their strong immunogenic function, is their capacity to migrate from sites of antigen capture to the draining lymphoid organs. Here we studied in detail the migratory pathway and the differentiation of DC during migration in a skin organ culture model and, for comparison, in the conventional contact hypersensitivity system. We report several observations on the capacity of cutaneous DC to migrate in mouse ear skin. (i) Upon application of contact allergens in vivo the density of Langerhans cells in epidermal sheets decreased, as determined by immunostaining for major histocompatibility complex class II, ADPase, F4/80, CD11b, CD32, NLDC-145/DEC-205, and the cytoskeleton protein vimentin. Evaluation was performed by computer assisted morphometry. (ii) Chemically related nonsensitizing or tolerizing compounds left the density of Langerhans cells unchanged. (iii) Immunohistochemical double-staining of dermal sheets from skin organ cultures for major histocompatibility complex class II and CD54 excluded blood vessels as a cutaneous pathway of DC migration. (iv) Electron microscopy of organ cultures revealed dermal accumulations of DC (including Birbeck granule containing Langerhans cells) within typical lymphatic vessels. (v) Populations of migrating DC in organ cultures upregulated markers of maturity (the antigen recognized by monoclonal antibody 2A1, CD86), but retained indicators of immaturity (invariant chain, residual antigen processing function). These data provide additional evidence that during both the induction of contact hypersensitivity and in skin organ culture, Langerhans cells physically leave the epidermis. Both Langerhans cells and dermal DC enter lymphatic vessels. DC mature while they migrate through the skin.

Langerhans cells require signals from both tumour necrosis factor-alpha and interleukin-1 beta for migration.

The induction phase of contact sensitization is associated with the movement of epidermal Langerhans cells (LC) from the skin and their migration, via afferent lymphatics, to draining lymph nodes where they accumulate as immunostimulatory dendritic cells (DC). It has been demonstrated previously that tumour necrosis factor-alpha (TNF-alpha) provides an important signal for LC migration and that in the absence of this cytokine, movement of LC from the epidermis to regional lymph nodes is inhibited. Recent evidence indicates that interleukin-1 beta (IL-1 beta), a cytokine produced in murine epidermis exclusively by LC, may also play a role in LC migration. The purpose of the investigations described here was to clarify, using relevant neutralizing anti-cytokine antibodies, the contributions made by TNF-alpha and IL-1 beta to the migration of LC from the epidermis. It was found that like anti-TNF-alpha, anti-IL-1 beta administered systemically to mice (by intraperitoneal injection), prior to skin sensitization with the contact allergen oxazolone, resulted in a marked inhibition of DC accumulation in draining lymph nodes. It was shown also that anti-IL-1 beta inhibited TNF-alpha-induced LC migration and DC accumulation and that; in similar fashion, the stimulation of LC migration and DC accumulation induced by IL-1 beta was compromised by prior treatment with anti-TNF-alpha. Based upon these data it is proposed that the stimulation of LC migration in response to skin sensitization requires the receipt by LC of two independent signals, one provided by TNF-alpha and the other by IL-1 beta. Morphological analyses of LC in epidermal sheets prepared from animals exposed to these cytokines with or without prior systemic treatment with anti-cytokine antibody suggested that the changes induced in LC by TNF-alpha and IL-1 beta may include the altered expression of adhesion molecules and acquisition of the ability to interact with and pass through the basement membrane.

Sun exposure, sunscreen and their effects on epidermal Langerhans cells.

This study examined the effects of chronic and current sun exposure on the number of Langerhans cells in epidermal sheets of UV-exposed and unexposed skin of the arms and assessed the effect of sunscreens. Participants were enrolled in a skin cancer prevention trial and had been using sunscreen daily for the previous 3 years. There were significantly fewer Langerhans cells on the exposed (463 cells/mm2) than on the unexposed forearm (528 cells/mm2) (P = 0.0001). High sun exposure in the previous 2 weeks and a history of predominantly outdoor occupations were both associated with a reduced number of Langerhans cells, although age and other biological indicators of chronic exposure were not associated. Sunscreen use was protective against the effects of current but not chronic sun exposure, with a suggestion of a greater effect at higher levels of exposure. Unexpectedly, people with a past history of nonmelanoma skin cancer had more Langerhans cells in both the exposed and the unexposed skin. These results emphasize the need for continued public health education to protect the immune system from the damaging effects of UV radiation.

Quantitative and three-dimensional analysis of human Langerhans cells in epidermal sheets and vertical skin sections.

We used confocal laser scanning microscopy to analyze and compare Langerhans cells (LCs) in normal skin of six subjects. Acetone-fixed epidermal sheets and 25-microns vertical skin sections were incubated with fluorescein isothiocyanate-conjugated mouse monoclonal antibodies directed against HLA-DR. An individual threshold setting algorithm compensating for the differences in background fluorescence was applied to identify specific fluorescence. No statistically significant difference was found in the relative volume of epidermal HLA-DR reactivity between epidermal sheets (14 +/- 5%) and vertical skin sections (13 +/- 6%) or in the number of dendrites per HLA-DR+ LCs (7.8 +/- 3.1 and 5.9 +/- 3.1, n = 58, respectively). However, statistically significant higher background intensity was found in vertical sections than in epidermal sheets. Three-dimensional (3D) reconstructions of HLA-DR+ LCs revealed a concentration of HLA-DR to one or a few intracellular vesicles in 42 of 58 analyzed LCs in epidermal sheets and in 18 of 58 analyzed LCs in vertical sections. Direct contact between dendrites from different LCs was not found. The results indicate that both skin forms are suitable for quantitative studies. Owing to less background intensity and larger tissue volume, detailed 3D analysis of LCs is preferably performed on epidermal sheets rather than on vertical sections.

Migration and maturation of Langerhans cells in skin transplants and explants.

The behavior of Langerhans cells (LC) has been examined after skin transplantation and in an organ culture system. Within 24 h (and even within 4 h of culture), LC in epidermal sheets from allografts, isografts, and explants dramatically increased in size and expression of major histocompatibility complex class II molecules, and their numbers were markedly decreased. Using a new procedure, dermal sheets were then examined. By 24 h, cells resembling LC were found close to the epidermal-dermal junction, and by 3 d, they formed cords in dermal lymphatics before leaving the skin. In organ culture, the cells continued to migrate spontaneously into the medium. These observations establish a direct route for migration of LC from the epidermis into the dermis and then out of the skin. These processes are apparently induced by a local inflammatory response, and are independent of host-derived mediators. The phenotype of migratory cells was then examined by two-color immunocytochemistry and FACS analysis. The majority of migratory leukocytes were Ia+ LC, the remainder comprised Thy-1+, CD3+, CD4-, CD8- presumptive T cell receptor gamma/delta+ dendritic epidermal cells, which clustered with the LC, and a small population of adherent Ia-, FcRII+, CD11a/18+ macrophages. In contrast to the cells remaining within the epidermis of grafted skin at 1 d, the migratory cells were heterogeneous in phenotype, particularly with respect to F4/80, FcRII, and interleukin 2 receptor alpha expression, which are useful markers to follow phenotypic maturation of LC. Moreover, cells isolated from the epidermis of grafts at 1 d were more immunostimulatory in the allogeneic mixed leukocyte reaction and oxidative mitogenesis than LC isolated from normal skin, though less potent than spleen cells. The day 1 migratory cells were considerably more immunostimulatory than spleen cells, and day 3-5 migratory cells even more so, suggesting that functional maturation continues in culture. Thus, maturation of LC commences in the epidermis and continues during migration, but the cells do not need to be fully mature in phenotype or function before they leave the skin. In vivo, the migration of epidermal LC via the dermis into lymphatics and then to the draining nodes, where they have been shown previously to home to T areas, would provide a powerful stimulus for graft rejection.

HLA antigens and Langerhans cell density in contact dermatitis.

We studied 67 patients with multiple contact allergies to determine whether there was an association of this state with any particular HLA antigen. HLA-A, -B and DR antigens were typed by standard serological methods. There was no significant HLA association, although there was an increased frequency of DR4 in those patients who included nickel as one of their sensitivities (64% compared with 33% in controls), and an increase in DR6 in those patients who included sensitivity to a rubber accelerator (45% compared with 16% in controls). However, when the probabilities were corrected for the number of HLA antigens tested and the number of substances in the patch test battery, these associations were no longer statistically significant. We also examined the morphology and numbers of Langerhans cells in epidermal sheets from six subjects with multiple allergies. There were no differences in appearance or numbers of Langerhans cells stained for ATPase, compared with 20 non-allergic controls.

The effects of ultraviolet light and certain drugs on Ia-bearing Langerhans cells in murine epidermis

Langerhans cells and indeterminate cells are immune macrophages of the epidermis and have Ia markers on their surface. Because of their position in the epidermis, they are subject to many environmental toxins like ultraviolet light. Also medications like cortisone applied topically to the skin could have important effects on these cells. We have used an anti-Ia serum and an indirect immunofluorescent technique to study Langerhans cells in epidermal sheets. We found that shortwave ultraviolet light (250–320 nm) and ultraviolet B (280–320nm) increased the density of Ia-bearing cells (Langerhans cells) in the skin. Psoralens and ultraviolet A (PUVA) (320–400 nm) depleted the skin of Ia-bearing cells, an effect which takes 2 weeks to produce but which persists for several weeks after stopping treatment. Triamcinolone acetonide administered topically or intraperitoneally also depletes the skin of Ia-bearing cells. These agents, light and steroids, either destroy the Ia-bearing cells or remove the Ia markers from the cellular surface.

A method for quantifying Langerhans cells in epidermal sheets of human and murine skin.

Langerhans cells are dendritic cells located in the epidermis. Several techniques have been used previously to stain these cells in sheets of epidermis separated from dermis. The ATPase technique is most commonly used but has several significant defects, the most important being that it is inconstant and nonspecific. Langerhans cells and indeterminate cells are the only dendritic cells in the epidermis which have Ia antigens on their surface. We have adapted an indirect immunofluorescence method to stain this population of cells in epidermal sheets. The technique is sensitive, specific, and can be used to identify and quantify Langerhans cells in human or murine epidermal sheets.