Pimecrolimus leads to an apoptosis-induced depletion of T cells but not Langerhans cells in patients with atopic dermatitis

BACKGROUND: Apart from the long-used corticosteroids, topical calcineurin inhibitors (tacrolimus, pimecrolimus) represent novel therapeutic options for the treatment of atopic dermatitis. OBJECTIVE: This study was designed to investigate the pathophysiological target cells and mode of action of pimecrolimus in atopic skin. METHODS: Twenty-two patients were randomly assigned to treatment with pimecrolimus cream 1%, matching vehicle cream, or beta-methasone-17-valerate (BMV) cream 0.1% in a randomized, double-blind, vehicle-controlled, parallel group clinical trial. Treatment was given twice daily for 3 weeks. Cryostat sections of skin biopsies were taken before as well as at selected time points after initiation of therapy. For certain experiments, healthy volunteers were topically treated with the creams described twice a day on 5 consecutive days. Epidermal sheets were prepared from suction blisters. For in vitro experiments, untreated, healthy human skin was obtained from patients undergoing plastic surgery. The effect of pimecrolimus and BMV on Langerhans cells (LCs), inflammatory dendritic epidermal cells, and T cells was investigated by using immunofluorescence and flow-cytometry techniques. RESULTS: While topical BMV treatment depleted LCs in healthy and atopic skin, pimecrolimus did not affect their number. Correlating with the disappearance of inflammatory cells, we observed a depletion of inflammatory dendritic epidermal cells and T cells on pimecrolimus and BMV treatment. Furthermore, we show that pimecrolimus depletes T cells by the induction of apoptosis. CONCLUSION: In summary, our data show that pimecrolimus reduces pathological T cells in atopic dermatitis skin via apoptosis, whereas LCs remain unaffected.     

Reduced dermal infiltration of cytokine-expressing inflammatory cells in atopic dermatitis after short-term topical tacrolimus treatment

BACKGROUND: In several clinical studies, topical calcineurin inhibitors have been shown to be effective in the treatment of atopic dermatitis (AD). They target signaling pathways that control gene expression, particularly the expression of cytokines. OBJECTIVE: We examined the cellular infiltrate in skin lesions of 10 patients with AD and characterized the cytokine pattern expressed by the infiltrating cells before and after short-term topical therapy with tacrolimus 1% ointment. METHODS: Skin biopsies were examined for histologic alterations (hematoxylin and eosin staining), composition of the cellular inflammatory infiltrate (immunofluorescence), and cytokine expression (ribonuclease protection assay, ELISA, immunofluorescence) before as well as 1 and 3 weeks after initiation of tacrolimus therapy. For comparison, biopsies from nonlesional AD and normal skin were analyzed. Systemic immunologic effects were assessed by analyzing peripheral blood leukocytes (immunofluorescence) as well as in vitro stimulated pan-T-cell cytokine production (ELISA). RESULTS: All patients showed a significant improvement of their skin lesions associated with a marked regression of spongiosis, acanthosis, and density of the cellular infiltrate in the dermis. The last was a result of reduced infiltration of T cells, B cells, and eosinophils. In contrast, the numbers of mast cells did not change. Moreover, the expression of the T H 2 cytokines IL-5, IL-10, and IL-13 in CD4 + T cells was reduced after therapy. Interestingly, tacrolimus therapy was also associated with a reduction of CD8 + T cells expressing the T H 1 cytokine IFN-gamma. Furthermore, the numbers of epidermal CD1a + dendritic cells increased after treatment. In the peripheral blood, a decrease of granulocytes (eosinophils and neutrophils) but no changes in the distribution of lymphocyte subpopulations were noticed. CONCLUSION: Topical tacrolimus treatment has anti-inflammatory effects on AD skin as indicated by reduced infiltration of cytokine expressing inflammatory cells. No evidence for drug-induced systemic immunosuppression was obtained.     

Immunological activation of dermal Langerhans cells in contact with lymphocytes in a model of human inflamed skin

Langerhans cells play an important role in the skin's immune system. Little is known, however, about the antigen-presenting capacity of Langerhans cells in the context of skin inflammation. By immunohistochemistry we investigated the phenotypic characteristics of epidermal and dermal Langerhans cells and their spatial relationship with infiltrating lymphocytes. We studied skin flaps autotransplanted to the oral cavity to fill a defect after maxillofacial cancer surgery. In 15 of 21 cases sampled for the present study, the skin flaps were severely inflamed by Candida albicans infection. In contrast to the normal skin, such inflamed skin showed a marked increase in CD1a(+) dermal Langerhans cells. Double immunohistochemistry revealed that dermal Langerhans cells abundantly expressed B7-2 (CD86), a representative costimulatory molecule, and CD83, a marker of mature dendritic cells. Furthermore, these dermal Langerhans cells were in close contact with CD4(+)/CD45RO(+) lymphocytes. This cell-to-cell contact was further visualized by immunoelectron microscopy. Langerhans cells were also observed within lymphatic vessels that were identified by the expression of vascular endothelial growth factor receptor-3. Ki-67 labeling indices were 4.2% in CD4(+) T cells and 0.8% in CD8(+) T cells within the dermis. Factor XIIIa(+) dermal dendrocytes were distributed outside the clusters of lymphocytes and were not in contact with them. Our observations indicate that dermal Langerhans cells in the inflamed skin are activated to express common phenotypes to mature dendritic cells so that they could stimulate neighboring memory CD4(+) T cells.     

Phenotypic transformation of macrophages to Langerhans cells in the skin

Langerhans cells, important participants in the cutaneous cellular immune response, are markedly diminished in skin of patients undergoing allogeneic bone marrow transplantation during the first 4 weeks after this procedure. To determine the mechanism responsible for the subsequent repopulation of these cells, the authors studied the immunophenotypic and morphologic profiles of sequential skin biopsies during the posttransplantation period. Cells with surface antigens of monocytes/macrophages within the superficial dermis were gradually replaced by dermal and epidermal dendritic cells exhibiting coexpression of monocyte/macrophage and Langerhans cell surface antigens. Ultrastructural examination revealed that many of these cells contained both prominent phagolysosomes and Birbeck granules. Antigenically and structurally mature Langerhans cells were observed within the epidermis by the end of the second month after transplantation. Phenotypic transformation of phagocytic dermal macrophages to Langerhans cells appears to represent a mechanism for repopulation of Langerhans cells during the period of immunologic reconstitution in this patient population.     

Thalidomide induces granuloma differentiation in sarcoid skin lesions associated with disease improvement

Sarcoidosis, a chronic granulomatous disease of unknown etiology, is treated with immune suppressive drugs such as corticosteroids. Sarcoidosis patients have been reported to benefit clinically from treatment with thalidomide. We administered thalidomide for 16 weeks to eight patients with chronic skin sarcoidosis and evaluated the drug's effects before and with treatment. After thalidomide treatment, all skin biopsies showed decreases in granuloma size and reduction in epidermal thickness. We also observed extensive T cell recruitment into the granulomas, the appearance of multinucleated giant cells, and increased numbers of dermal Langerhans cells (CD1a(+)) and mature dendritic cells (CD83(+) or DC-LAMP(+)). Plasma IL-12 levels increased and remained elevated during the treatment period. We noted increased HLA-DR expression on peripheral blood lymphocytes and a corresponding drop in the naive T cell marker CD45RA. Our data suggest that thalidomide treatment of sarcoidosis results in granuloma differentiation to a Th1-type cellular immune response usually associated with protective immunity to tuberculosis and tuberculoid leprosy.     

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.     

Immunohistologic assessment of cytokine production of infiltrating cells in various forms of leprosy.

The aim of this study was to determine cytokines in human leprosy lesions by means of immunohistologic examination. Cryostat sections of skin biopsies from 57 patients with various forms of leprosy were immunostained according to the APAAP method, using monoclonal antibodies against interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and, in addition, against CD 1 antigen. Granulomas in biopsies of untreated patients with tuberculoid leprosy showed large amounts of cells positive for IL-1 beta, TNF-alpha, IFN-gamma, and CD 1, whereas no positive signals could be detected in untreated patients with lepromatous leprosy. However, in those biopsies obtained from lepromatous leprosy patients undergoing chemotherapy, positive staining for cytokines as well as subepidermal Langerhans cells increased to a detectable amount. Remarkably, in tuberculoid leprosy patients, the number of IL-1 beta--positive cells did not vary under therapy, while the number of TNF-alpha and IFN-gamma reactive cells decreased. These results suggest that immunohistologic determination of cytokines in combination with the assessment of subepidermal Langerhans cells in human leprosy lesions may be used as a parameter for the patient's status of cell-mediated immunity under chemotherapeutic treatment.     

Development and maturation of Langerhans cells, spleen and bone marrow dendritic cells in TNF-alpha/lymphotoxin-alpha double-deficient mice

Dendritic cells are key regulators of immunity and tolerance. TNF-alpha has manifold effects on dendritic cells. It is an indispensable ingredient in several dendritic cell generation protocols, especially in the human, and it is included in diverse maturation stimuli for dendritic cells. Mice deficient in various components of the TNF/lymphotoxin system (TNF-alpha, lymphotoxin-alpha and -beta, TNF receptors, combinations thereof) have profound defects in mounting immune responses to infections. The dendritic cell system in these mice has been incompletely studied to date. We therefore investigated dendritic cells from the epidermis (Langerhans cells), spleen and the bone marrow of mice double-deficient in TNF-alpha and lymphotoxin-alpha. We report that dendritic cells in these mice are grossly normal. Langerhans cells, spleen and bone marrow dendritic cells can develop and mature. Their expression of MHC II and CD86 is not impaired, and their T cell-stimulatory as well as antigen-processing capacity is comparable to their normal counterparts. Thus, the described defects in these mice appear to be due the lack of lymph nodes, the disturbed architecture of the spleen, and deranged chemokine production patterns, rather than to a profoundly altered dendritic cell system.     

Immunohistochemical study of epidermal Langerhans cells and dermal dendritic cells in benign and malignant skin lesions characterized by a dermal lymphoid infiltrate consisting either of B-cells or T-cells

Summary Skin biopsies from 43 patients with a rather dense dermal lymphoid infiltrate of either inflammatory or neoplastic nature have been investigated. We studied the number, distribution and immunophenotype of epidermal Langerhans cells and dermal dendritic cells. As previously reported, differences in epidermal Langerhans cell and dermal dendritic cell numbers between skin biopsies with a B-cell infiltrate and skin biopsies with a T-cell infiltrate were found, dendritic cells being more numerous in the latter. The main finding of this study was an uneven distribution of epidermal Langerhans cells and dermal dendritic cells in skin biopsies with a T-cell infiltrate: in skin lesions with an inflammatory lymphoid infiltrate, small clusters of epidermal and dermal dendritic cells admixed with T-lymphocytes (predominantly T-helper/inducer cells) and small blood vessels were present at areas of exocytosis. In skin lesions with a neoplastic lymphoid infiltrate larger, more loosely arranged aggregates of dendritic cells and T-cells were seen. These cell aggregations composed of activated (inflammatory or neoplastic) T-cells and dendritic cells may represent the cutaneous homologue of the secondary T-nodule in the lymph node. Both types of cell aggregates may correspond to the dendritic cell-T cell clusters observed in in vitro induced immune responses.Presented at the XVIth International Congress of the International Academy of Pathology and 7th World Congress of Academic and Environmental Pathology in Vienna, 1986Aspirant of the NFWO (Nationaal Fonds voor Wetenschappelijk Onderzoek)     

Distribution of ICAM-3-bearing cells in normal human tissues. Expression of a novel counter-receptor for LFA-1 in epidermal Langerhans cells.

The LFA-1 integrin mediates its function in leukocyte intercellular interactions by recognition of at least one of its three identified counter-receptors, intercellular adhesion molecules 1 (ICAM-1), 2, and 3. The ICAM-1 molecule is expressed in an inducible-dependent manner by endothelial and epithelial cells, as well as by other cell types, including leukocytes. On the other hand, ICAM-2 is constitutively expressed mainly by endothelial cells. We have studied the tissue distribution of the ICAM-3 molecule by immunohistochemical staining of lymphoid and nonlymphoid organs. Only cells from the leukocyte lineage were found bearing the ICAM-3 antigen, showing a pattern of expression clearly distinct from those of ICAM-1 and ICAM-2. Interestingly, we have found that ICAM-3 is expressed by epidermal dendritic Langerhans cells as assessed by double immunostaining with antibodies specific for CD1. In contrast, staining of skin sections with anti-ICAM-1 and ICAM-2 antibodies showed an undetectable expression of these two molecules on Langerhans cells. However, CD1+ Langerhans cells localized in the paracortical area of dermatopathic lymph nodes expressed both ICAM-1 and ICAM-3 antigens. Our results indicate that ICAM-3 is the main LFA-1 counter-receptor in human resident epidermal Langerhans cells. ICAM-3 may have an important role in T-cell antigen stimulation driven by Langerhans cells during skin immune reactions.     

Psoriasis in humans is associated with down-regulation of galectins in dendritic cells

We have investigated the expression and role of galectin‐1 and other galectins in psoriasis and in the Th1/Th17 effector and dendritic cell responses associated with this chronic inflammatory skin condition. To determine differences between psoriasis patients and healthy donors, expression of galectins was analysed by RT‐PCR in skin samples and on epidermal and peripheral blood dendritic cells by immunofluorescence and flow cytometry. In the skin of healthy donors, galectin‐1, ‐3 and ‐9 were expressed in a high proportion of Langerhans cells. Also, galectins were differentially expressed in peripheral blood dendritic cell subsets; galectin‐1 and galectin‐9 were highly expressed in peripheral myeloid dendritic cells compared with plasmacytoid dendritic cells. We found that non‐lesional as well as lesional skin samples from psoriasis patients had low levels of galectin‐1 at the mRNA and protein levels, in parallel with low levels of IL‐10 mRNA compared with skin from healthy patients. However, only lesional skin samples expressed high levels of Th1/Th17 cytokines. The analysis of galectin‐1 expression showed that this protein was down‐regulated in Langerhans cells and dermal dendritic cells as well as in peripheral blood CD11c⁺ DCs from psoriasis patients. Expression of galectin‐1 correlated with IL‐17 and IL‐10 expression and with the psoriasis area and index activity. Addition of galectin‐1 to co‐cultures of human monocyte‐derived dendritic cells with autologous T lymphocytes from psoriasis patients attenuated the Th1 response. Conversely, blockade of galectin binding increased IFNγ production and inhibited IL‐10 secretion in co‐cultures of monocyte‐derived dendritic cells with CD4⁺ T cells. Our results suggest a model in which galectin‐1 down‐regulation contributes to the exacerbation of the Th1/Th17 effector response in psoriasis patients. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Activated Langerhans cells release tumor necrosis factor

Langerhans cells act as antigen-presenting cells in immune reactions in the skin. What other roles they may play in inflammation is less well defined. We have tested whether these cells can produce TNF-alpha, an important mediator of inflammation. Resting Langerhans cells produce less than 0.1 U TNF-alpha/ml. Langerhans cells stimulated with phorbol myristate acetate (PMA) and lipopolysaccharide (LPS) release 4-5 U TNF-alpha/ml. Specificity of the released TNF-alpha in an L929 cytotoxicity assay was confirmed by using neutralizing anti-TNF-alpha monoclonal antibodies, and the identity of TNF-alpha was further confirmed by Northern blot hybridization with an TNF-alpha oligomer DNA probe. Activated Langerhans cells may contribute to inflammation in the skin by releasing TNF-alpha, which is known to effect fibroblast growth, endothelial cell activation, and lymphocyte function.     

Treatment with tumor necrosis factor-alpha and granulocyte-macrophage colony-stimulating factor increases epidermal Langerhans' cell numbers in cancer patients

Dendritic cells (DCs) initiate primary and stimulate secondary T-cell responses. We conducted a phase I trial of tumor necrosis factor (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in patients with cancer to increase DCs in peripheral blood or skin based on in vitro data that showed that CD34(+) hematopoietic precursors require these cytokines to mature into functional antigen-presenting DCs. Eleven patients were treated for 7 days with GM-CSF, 125 microg/m(2) twice daily as subcutaneous injections, and TNF-alpha as a continuous infusion at dose levels of 25, 50, or 100 microg/m(2)/day. The maximum tolerated dose of TNF-alpha was 50 microg/m(2)/day with this dose of GM-CSF; dose-limiting toxicities occurred in both patients treated with 100 microg/m(2)/day. One became thrombocytopenic and the other had transient confusion. Epidermal Langerhans' cells were quantitated by S100 staining of skin biopsies and DC precursors in peripheral blood by colony-forming unit dendritic (CFU-dendritic) assays. S100-positive cells in the epidermis doubled after treatment (2.55 S100(+) cells/high-power field before treatment to 6.05 after treatment, p = 0.029). CFU-dendritic in peripheral blood increased after treatment in 3 colorectal cancer patients but not in 3 patients with melanoma. CD11c(+) or CD123(+), HLA-DR(bright), lineage-negative dendritic cell precursors were not increased in peripheral blood mononuclear cells. This trial demonstrates that treatment with TNF-alpha and GM-CSF can increase the number of DCs in the skin and the number of dendritic cell precursors in the blood of some patients with cancer. This approach may increase the efficacy of vaccination to tumor antigens in cancer patients.     

Origin and migratory properties of dendritic cells in the skin

The professional antigen presenting cells of the skin, namely Langerhans cells and dermal dendritic cells, are continuously exposed to environmental stimuli. The population of antigen-carrying dendritic cells that migrates from this site to the draining lymph nodes is highly dynamic, and its density, composition and function changes as a consequence of dendritic cell recruitment and activation in tissues, as well as interactions with T lymphocytes in the specialized lymph node microenvironments. Therefore dendritic cells within the skin are the key regulators of both quantitative and qualitative aspects of immune responses to local antigenic challenge.     

Interaction of lipoteichoic acid and CpG-DNA during activation of innate immune cells

The innate immune system recognizes pathogen-associated molecular patterns (PAMP) to cope with evolving infections. Toll-like receptors (TLRs) play a pivotal role in recognition of PAMPs. In the course of infection not a single but rather a full panel of different microbial components interacts with distinct TLRs simultaneously. Only limited information is available on effects of combinations of TLR agonists. Here, we have analyzed the effects of lipoteichoic acid (LTA), CpG-DNA and combinations thereof on innate immune cells in vitro. Although proinflammatory cytokines like TNF-alpha were induced by these agonists in quite similar amounts, CpG DNA was superior in its potency to induce IL-12p40 reflecting important differences in the biological valence of LTA and CpG-DNA. When given in combination, LTA and CpG-DNA were additive in induction of TNF-alpha, IL-6 and nitric oxide in RAW 264 macrophages, peritoneal macrophages and dendritic cells. Additive effects were also observed in regard to TNF-alpha mRNA. In contrast, LTA suppressed IL12p40 secretion induced by CpG-DNA in RAW cells and peritoneal macrophages but not in dendritic cells. Intracellular signal cascades (NFkappaB and p38 MAP kinase) showed additive effects after simultaneous triggering. mRNA expression ofTLRs showed only minor regulation after CpG or LTA application and thus does not account for the additive/suppressive effects observed. These results indicate that the consequences of interaction of innate immune cells with microbial pattern depend on the responding cell type and might be differential for certain effector mechanisms. Thus, the pathogen-characteristic panel of TLR ligands will induce pathogen-specific innate responses decisive for the inflammatory reactions.     

Localization of tumour necrosis factor-alpha (TNF-alpha) and its receptors in normal and psoriatic skin: epidermal cells express the 55-kD but not the 75-kD TNF receptor.

The distribution of TNF-alpha, p55 TNF receptor (TNF-R) and p75 TNF-R in normal skin and uninvolved and lesional skin from psoriasis patients has been investigated, using specific mono- and polyclonal antibodies. In normal skin, and uninvolved and lesional skin from psoriasis patients, p55 TNF-R is associated with epidermal keratinocytes and a network of upper dermal dendritic cells. This suggests that the actions of TNF-alpha on epidermal cells in vivo are mediated by binding to the p55 TNF-R. In lesional psoriasis skin, there was staining of the parakeratotic stratum corneum and increased expression of p55 TNF-R in association with upper dermal blood vessels. Staining for p75 TNF-R in normal skin was restricted to eccrine sweat ducts and dermal dendritic cells, and was absent from the epidermis. In lesional psoriasis skin, there was staining for p75 TNF-R in association with upper dermal blood vessels and perivascular infiltrating cells. TNF-alpha in normal skin was predominantly localized to the basal cell layers of the epidermis, and was seen in association with eccrine ducts and sebaceous glands. In lesional psoriasis skin, and to a lesser extent in uninvolved psoriasis skin, TNF-alpha was distributed throughout the epidermis, and was also specifically localized to upper dermal blood vessels. Up-regulation of TNF-alpha, p55 TNF-R and p75 TNF-R on dermal blood vessels in psoriasis may play an important role in the pathogenesis of this condition by promoting cutaneous recruitment of inflammatory cells.     

Large-scale isolation of immature dendritic cells with features of Langerhans cells by sorting CD34+ cord blood stem cells cultured in the presence of TGF-beta1 for cutaneous leukocyte antigen (CLA)

Epidermal Langerhans cells (LCs) are a subset of immature dendritic cells (DCs) and play a key role in the initiation and regulation of T cell responses. Upon antigenic stimulation, LCs differentiate into mature DCs undergoing profound morphologic and functional changes. Studies of the biological details of this conversion process have been hampered by difficulties in generating immature dendritic cells of a defined lineage. We propose a new method of purifying homogenous immature DCs in large numbers by sorting for CLA (Langerhans-like cells) from cord-blood-derived haematopoietic progenitor cells (HPCs). Established protocols describe the generation of LCs from CD34(+) HPCs by sorting for CD1a after 5 days of culture in the presence of GM-CSF and TNF-alpha. However, the numbers of LCs obtained by this method remain within the low range. Furthermore, CD1a is also expressed on interstitial DCs. LCs but not interstitial DCs express the cutaneous leukocyte antigen (CLA). The expression of CLA by cells stimulated with TNF-alpha and GM-CSF peaks on day 10. This expression can be raised further by stimulating the cells with TGF-beta1 and omitting TNF-alpha from day 6 onwards. CLA(+) cells were isolated on day 10 by AutoMACS. Their LC phenotype was established by the presence CD207. The immaturity of Langerhans-like cells was shown by the lack of CD83 and CD208 expression as well as their lower ability to activate allogeneic naive T cells as compared to maturing dendritic cells. However, CLA(+) cells cannot be termed Langerhans cells as they do not express Birbeck granules. Compared to sorting for CD1a (on day 6), sorting for CLA (on day 10) results in isolates of higher purity (80% vs. 50%) and a yield eight times higher (4.9x10(6) vs. 6.5x10(5) cells) when using identical numbers of input cells (5x10(5) cells). This novel method guarantees large numbers of pure and functionally active immature dendritic cells.     

Extraction of human Langerhans cells: a method for isolation of epidermis-resident dendritic cells

Langerhans cells (LCs) are immature dendritic cells in the epidermis that play a central role in T-lymphocyte mediated skin immunity. Upon activation with antigenic stimuli, they differentiate drastically into mature dendritic cells while migrating from the epidermis to regional lymph nodes. Thus, in order to study biological details of immature LCs, it is crucial to isolate epidermis-resident, immature LCs without dermal dendritic cell contamination. Methods for extracting LCs from human skin as well as in vitro derivation of LC-like cells from hematopoietic progenitor cells have been described previously, but the cell preparations can potentially contain a significant number of dendritic cells that are not identical to epidermal LCs. Here, we describe a technique by which purely epidermis-resident LCs are extracted from human skin. Following digestion of human skin with dispase, the epidermis was separated mechanically without any attached dermal component. The trypsinized epidermal cells were then fractionated by centrifugation with a discontinuous density gradient composed of bovine albumin and sodium metrizoate. The LC-enriched preparation thus obtained contained 80% to >90% CD1a+, E-cadherin+ cells that expressed Birbeck granules and the Lag protein. Consistent with their being at an immature stage, the freshly isolated LCs lacked the expression of CD83, a marker for mature dendritic cells. The purified LCs were able to activate allogeneic T cells, indicating that the cells retained T-cell stimulation ability even after extraction. Thus, the present work offers an opportunity for precise in vitro studies of epidermal LCs.     

Langerhans cell depletion in gliotoxin-treated murine epidermis.

Langerhans cells (LC) are dendritic antigen presenting cells of bone marrow origin which reside in the suprabasal layer of the epidermis. They express high concentrations of Class II MHC glycoproteins on their plasma membrane and transport cutaneous antigen to local lymph nodes for presentation to helper T cells. They are thus essential for the induction of cutaneous immunity. Gliotoxin is a member of the epipolythiodioxopiperazine (ETP) group of fungal metabolites, derived from the human pathogen Aspergillus fumigatus. It has been shown to have immunomodulating properties in vivo and in vitro, and has been proposed as a potential immunosuppressant for transplantation therapy. Epicutaneous application of gliotoxin reduced the numbers of epidermal LC by 30-35 per cent with an associated morphological change from highly dendritic to a more rounded form. Electron microscopic studies showed selective damage to LC at very low (nM) concentrations of gliotoxin, with no obvious effect on adjacent keratinocytes. LC numbers remained depleted for 13 weeks after initial treatment, suggesting that systemic suppression or prolonged retention of gliotoxin within the skin may play a role in its mechanism of action.     

Characterization of Canine Dendritic Cells in Healthy,Atopic, and Non-allergic Inflamed Skin

Atopic dermatitis in humans and dogs is a chronic relapsing allergic skin disease. Dogs show a spontaneous disease similar to the human counterpart and represent a model to improve our understanding of the immunological mechanisms, the pathogenesis of the disease, and new therapy development. The aim of the study was to determine the frequency and phenotype of dendritic cells (DC) in the epidermis and dermis of healthy, canine atopic dermatitis lesional, and non-allergic inflammatory skin to further validate the model and to obtain insights into the contribution of DC to the pathogenesis of skin diseases in dogs. We first characterized canine skin DC using flow-cytometric analysis of isolated skin DC combined with an immunohistochemical approach. A major population of canine skin dendritic cells was identified as CD1c⁺CD11c⁺CD14⁻CD80⁺MHCII⁺MAC387⁻ cells, with dermal DC but not Langerhans cells expressing CD11b. In the epidermis of lesional canine atopic dermatitis and non-allergic inflammatory skin, we found significantly more dendritic cells compared with nonlesional and control skin. Only in canine atopic dermatitis skin did we find a subset of dendritic cells positive for IgE, in the epidermis and the dermis. Under all inflammatory conditions, dermal dendritic cells expressed more CD14 and CD206. MAC387⁺ putative macrophages were absent in healthy but present in inflamed skin, in particular during non-allergic diseases. This study permits a phenotypic identification and differentiation of canine skin dendritic cells and has identified markers and changes in dendritic cells and macrophage populations related to allergic and non-allergic inflammatory conditions. Our data suggest the participation of dendritic cells in the pathogenesis of canine atopic dermatitis similar to human atopic dermatitis and further validate the only non-murine spontaneous animal model for this disease.