A novel monoclonal antibody to a distinct subset of cutaneous dendritic cells.

A monoclonal antibody was generated by immunizing rats with Langerhans cell (LC)-enriched epidermal cells obtained from BALB/c mouse earskin after epicutaneous application of the contact sensitizer 2,4-dinitrofluorobenzene (DNFB). The antibody 4F7 detects in normal mouse skin, few dermal cells showing the morphologic, phenotypic, and functional properties of accessory dendritic cells, but lacking Birbeck granules. The capacity to stimulate allogenic T cells in the mixed leucocyte reaction resembles that of freshly isolated LCs. After DNFB application, an increased number of 4F7+ dendritic cells are found in the dermis and, in addition, some labeled dendritic cells occur in the epidermis. Some of the latter cells exhibit cytoplasmic Birbeck granules. Remarkably, there is no increase of the 4F7+ cells in the regional lymph nodes after DNFB treatment. These data suggest that the 4F7 antibody labels distinct dendritic cells of the mouse skin that are involved in the mediation of contact sensitization and probably represent immature LCs.     

Expression and function of the mannose receptor CD206 on epidermal dendritic cells in inflammatory skin diseases

The capability to take up mannosylated protein antigens is important for the biologic function of dendritic cells, as many glycoproteins derived from bacteria and fungi, e.g., Malassezia furfur, are mannosylated. The expression of the mannose receptor CD206 has been regarded a differentiation hallmark of immature dendritic cells, whereas monocytes and mature dendritic cells as well as epidermal Langerhans cells do not express CD206. This study describes some epidermal dendritic cells that may express CD206 under inflammatory skin conditions: Immunohistochemical and flow cytometric analysis with the CD206-specific D547 antibody confirmed that Langerhans cells from normal human skin do not express CD206. Epidermal cell suspensions from atopic dermatitis and psoriasis revealed two distinct subsets of epidermal dendritic cells: a CD1a(+++)/CD206(-) cell population (i.e., Langerhans cells) and a CD1a(+)/CD206(++) cell population, corresponding to the previously described inflammatory dendritic epidermal cells. CD206-mediated endocytosis, assessed by dextran-fluorescein isothiocyanate uptake, was demonstrated in inflammatory dendritic epidermal cells but not in Langerhans cells. CD206-independent uptake of the fluorescent dye Lucifer yellow, a pinocytosis marker, was demonstrated in both Langerhans cells and inflammatory dendritic epidermal cells. Electron microscopic examination, known to distinguish Langerhans cells from inflammatory dendritic epidermal cells by their Birbeck granules, revealed Langerhans cells with Birbeck granules and inflammatory dendritic epidermal cells without Birbeck granules. Inflammatory dendritic epidermal cells exhibited numerous coated pits and vesicles, the latter fusing with large endosome-like structures, thus suggesting a high endocytotic activity. Immunogold staining with D547 monoclonal antibody confirmed that inflammatory dendritic epidermal cells were positive for CD206. In conclusion, inflammatory dendritic epidermal cells but not Langerhans cells are expressing CD206 in situ and use it for receptor-mediated endocytosis.     

Skin irritants and contact sensitizers induce Langerhans cell migration and maturation at irritant concentration

Skin irritants and contact allergens reduce the number of Langerhans cells (LCs). It has been assumed that this reduction is due their migration to the draining lymph node (LN) for initiating immune sensitization in a host. Skin irritation, however, as opposed to contact allergy is not considered to be an immunological disease. Nevertheless, skin irritants are also known for their adjuvant-like effects on contact allergy, resulting in skin hypersensitivity reactions like toxic dermatitis. The human organotypic skin explant culture (hOSEC) model is used to study the characteristics of chemical-induced migration of CD1a(+) LCs out of the epidermis in relation to irritancy or toxicity. We analysed cells emigrating out of hOSEC for CD1a(+) LCs, CD83(+) mature dendritic cells (DCs) and CCR7(+) LN homing cells. After exposure to a toxic concentration of a non-immunogenic irritant, an increase of CD1a(+)CD83(+) LCs was found in the culture medium. A non-toxic concentration of an sensitizer induced an increase of CD1a(+) cells. About 50% of skin emigrating CD1a(+) LCs were CD83(-) (immature) but all were CCR7(+). Skin irritation by both non-allergenic and allergenic compounds induces LC migration and maturation. In contrast, only allergenic compounds induced LC migration with partial maturation at subtoxic concentration. This effectively demonstrates that irritation is physiologically needed stimuli for inducing LC maturation.     

Heterogeneity of dermal OKT6+ cells in inflammatory and neoplastic skin diseases

This immunopathologic study of both normal and pathologic skin specimens (contact dermatitis [CD], lichen planus [LP], cutaneous T cells lymphoma [CTCL], and histiocytosis X [HX]) allowed as to differentiate four types of dermal OKT6+ cells: (1) cells with the same morphologic features as epidermal Langerhans cells (LCs), rarely found in either normal or pathologic dermis; (2) cells structurally similar to LCs but lacking Birbeck granules (BGs), found mainly in CD and LP; (3) larger cells rich in cytoplasmic organelles, only 5% of which contained BGs. They were especially common CTCL; and (4) cells typical of HX.     

The presence of IgE molecules on epidermal langerhans cells in patients with atopic dermatitis

Summary Skin sections of clinically involved and clinically normal-looking skin from patients with atopic dermatitis were incubated with anti-human IgE antibodies using the indirect immunoperoxidase technique. Apart from positive dermal anti-IgE staining, positive epidermal anti-IgE staining was also observed. The morphology of the epidermal staining cells suggested the involvement of dendritic cells. This was confirmed by positive immuno-double labelling with OKT6 and anti-IgE. This phenomenon seemed to be specific for atopic dermatitis since skin sections from normal nonatopic controls, patients with allergic asthma, contact dermatitis, and schistosomiasis showed no epidermal anti-IgE staining. To further elucidate the nature of the epidermal anti-IgE staining cells, epidermal cell suspensions were prepared from clinically involved skin from patients with atopic dermatitis. These cell suspensions also showed positive anti-IgE staining cells and positive immuno-double labelling with OKT6 and anti-IgE. Immunogold electron microscopy with anti-IgE on epidermal cell suspensions from patients with atopic dermatitis showed gold particles on the cell membranes of cells containing Birbeck granules, being Langerhans' cells. Epidermal cell suspensions from normal non-atopic controls were negative. The presence of IgE molecules on epidermal Langerhans' cells, which seems to be specific for patients with atopic dermatitis, provides an explanation for the high frequency of positive patch test reactions to inhalant allergens.     

Cytochemical and Ultrastructural Studies of the Langerhans'' cells

In the guinea pig, experimental allergic contact dermatitis (ACD) and primary irritant contact dermatitis (PICD) were induced with different concentrations of dinitrochlorobenzene (DNCB). The epidermal Langerhans' cells (LCs) were observed sequentially by both adenosine triphosphatase (ATPase) and electron microscopy. Light microscopically, in ACD, the density and dendritic processes of LC decreased markedly within 12 h after antigen challenge. Almost no recognization LCs could be seen within 2 to 5 days. Later, LCs began to repopulate in the epidermis. Within 14 days, the density and shape of the LCs returned to normal. On the contrary, LCs changed more rapidly in PICD. The dendritic processes of LC decreased within 2 h and cell density decreased dramatically within 6 h after DNCB application. LCs also repopulated more rapidly in the epidermis. Electron microscopically, in ACD, we observed that lymphocyte-like cells apposed to LCs; LCs were activated and damaged; however, in PICD, we found neither the apposition of lymphocyte-like cells to LCs, nor the activation of LCs. LCs play an important role in the convalescence phase as well as in the early and later phases of contact allergic reaction.     

Electron microscopic study on Langerhans cells and related cells in lymph nodes of DNCB-sensitive mice

Summary To understand contact hypersensitivity, it is important to know the kinetics of Langerhans cells (LC) and related cells in the lymph node (LN), as well as in the skin. For this purpose, we tried experimentally to induce increased numbers of LCs, Birbeck granule-like structure (BgS)-containing cells, and interdigitating reticulum cells (IDC) in DNCB-sensitive mice and studied them by means of electron microscopy with the following results: (1) cytologically, LC, BgS-containing cells and IDC were closely related; (2) BgS seemed to arise from rough-surfaced endoplasmic reticulum (r-ER), and BgS-containing cells were midway in nature between LC and IDC from the morphological view point. From these findings, it appears that IDC, BgS-containing cells, and LCs were simultaneously involved in the contact hypersensitivity reactions of LNs.Abbreviations Al ethanol - Bg Birbeck granule - BgS Birbeck granulelike structure - DNCB 2,4-dinitro-1-chlorobenzene - IDC interdigitating reticulum cells - LC Langerhans cell - LN lymph node - r-ER rough-surfaced endoplasmic reticulum     

Cytochemical and Ultrastructural Studies of the Langerhans' Cells

Abstract: In the guinea pig, experimental allergic contact dermatitis (ACD) And primary irritant contact dermatitis (PICD) were induced with different concentrations of dinitrochlorobenzene (DNCB). The epidermal Langerhans' cells (LCs) were observed sequentially by both adenosine triphosphatase (ATPase) and electron microscopy. Light microscopically, in ACD, the density and dendritic processes of LC decreased markedly within 12 h after antigen challenge. Almost no recognization LCs could be seen within 2 to 5 days. Later, LCs began to repopulale in the epidermis. Within 14 days, the density and shape of the LCs returned to normal. On the contrary, LCs changed more rapidly in PICD. The dendritic processes of LC decreased within 2 h and cell density decreased dramatically within 6 h after DNCB application. LCs also repopulated more rapidly in the epidermis. Electron microscopically, in ACD, we observed that lymphocyte-like cells apposed to LCs; LCs were activated and damaged; however, in PICD, we found neither the apposition of lymphocyte-like cells to LCs, nor the activation of LCs. LCs play an important role in the convalescence phase as well as in the early and later phases of contact allergic reaction.     

TNF-α, RANTES, and MCP-1 are major chemoattractants of murine Langerhans cells to the regional lymph nodes

We have previously reported that lymph node cells generated chemotactic factors for Langerhans cells (LCs) in the induction phase of contact dermatitis. In order to clarify the chemotactic factors involved in migration to the regional lymph nodes, we investigated the migratory activity of murine LCs toward several cytokines and chemokines in vitro . One-day cultured LC-enriched epidermal cells were added to the upper compartment of a modified Boyden chamber and cytokines were added to the lower compartment. We counted dendritic cells migrated to the lower chamber as LCs under phase contrast microscopy. About 99% of migrated dendritic cells were positively reacted with anti-Ia^d and NLDC145 antibodies and considered to be LCs. We could detect LC migration more accurately by this direct examination than by counting the migrated cells in the filter membrane of a Boyden chamber. In our system, migration of murine epidermal LCs was stimulated by TNF-β, RANTES and MCP-1, but not by GM-CSF, IL-1β, IL-4, and IL-6. TNF-α induced LC migration at concentrations from 4X10³ U/ml to 5X10⁴ U/ml. RANTES at concentrations from 10 to 100 ng/ml and MCP-1 a concentration of 100 ng/ml induced LC migration in a dose-dependent manner. These data confirmed that TNF-α, RANTES, and MCP-1 induced LC migration from epidermis during contact sensitization.     

Enhanced expression of the high-affinity receptor for IgE (Fc(epsilon)RI) associated with decreased numbers of Langerhans cells in the lesional epidermis of atopic dermatitis

Epidermal Langerhans cells (LCs) and the high-affinity receptor for IgE (Fc(epsilon)RI) on their surface are considered important in the pathogenesis of atopic dermatitis (AD). We investigated the numbers of epidermal LCs and their Fc(epsilon)RI expression in patients with AD and healthy controls. Biopsy specimens taken from lesional skin from 17 patients with AD, non-lesional skin from five patients with AD and normal skin from five healthy individuals were immunohistochemically stained with a monoclonal antibody against CD1a or with either of two monoclonal antibodies against two different epitopes of Fc(epsilon)RI alpha chain. Many dendritic cells were positively stained with anti-CD1a antibody in the epidermis of each skin sample, and fewer cells were stained with anti-Fc(epsilon)RI antibodies. The numbers of epidermal LCs positive for Fc(epsilon)RI were significantly increased in both lesional and non-lesional skin from AD patients compared with those in normal skin, suggesting important roles of Fc(epsilon)RI+LCs in the pathogenesis of the disease. In contrast, the numbers of total epidermal LCs (CD1a-positive) were decreased in AD lesional skin compared with those in non-lesional skin from AD patients and in normal skin from healthy subjects. Together with our finding that the numbers of epidermal LCs were negatively correlated with the clinical severity of the AD lesions, we concluded that epidermal LCs may decrease in some conditions of AD, probably in lesions with severe inflammation.     

Down-regulation of Langerhans cell protein kinase C-β isoenzyme expression in inflammatory and hyperplastic dermatoses

The family of protein kinase C (PKC) isoenzymes plays a fundamental part in signal transduction, and thereby regulates important cellular functions, including growth, differentiation, cytokine production and adhesion molecule expression. In lesional psoriatic skin. Ca²⁺-dependent PKC activity, PKC-β protein and epidermal Langerhans cell (LC) PKC-β immunostaining are significantly decreased, indicating activation and subsequent down-regulation of PKC. Whether these changes occur in other inflammatory/hyperplastic dermatoses is, however, unknown. We examined PKC-α and PKC-β expression in normal skin, psoriasis, cutaneous T-cell lymphoma (CTCL), lamellar ichthyosis, non-bullous ichthyosiform erythroderma, atopic dermatitis, urushiol-induced allergic contact dermatitis, and sodium lauryl sulphate (SLS)-induced irritant contact dermatitis. Cryostat sections were stained for PKC-α and PKC-β, and the LC marker CDla, using an immunoperoxidase technique and specific monoclonal antibodies. Double-labelling studies, in normal skin, revealed co-expression of PKC-β and CDla by epidermal LCs. Analysis of the number of PKC-^β+ and CDl^a+ epidermal LCs, in diseased compared with normal skin, revealed three categories: (i) in psoriasis and CTCL. the PKC-^β+ epidermal LC number was significantly reduced, whereas the CDl^a+ epidermal LC number was unchanged; (ii) in allergic and irritant contact dermatitis, both PKC-β+ and CDla⁺ epidermal LCs were significantly reduced in number; and (iii) in atopic dermatitis, the PKC-β⁺ epidermal LC number was normal, and CDla⁺ epidermal LCs were significantly increased in number. Moreover, the ratio of epidermal LC PKC⁺/CDla⁺ was reduced in all the dermatoses studied, suggesting activation of PKC-β, with subsequent down-regulation. Within the dermis, increased PKC-β staining of infiltrating cells was observed in all the conditions studied except lamellar ichthyosis and non-bullous ichthyosiform erythroderma. These data indicate that: (i) down-regulation of LC FKC-β occurs in a variety of inflammatory and hyperplastic skin disorders, and is not unique to psoriasis, and (iii the pattern of epidermal LC PKC-β and CDla expression varies among the diseases studied. In mice, PKC activation induces LC migration. Thus, down-regulation of epidermal LC PKC-β associated with reduced CDla⁺ epidermal LCs in allergic and irritant contact dermatitis suggests that PK.C-β may transduce the signal for migration of LCs from human epidermis.     

The steady-state turnover of murine epidermal Langerhans cells

We have investigated the steady-state turnover of murine epidermal Langerhans cells (LCs) using an X-irradiation model, 3H-thymidine autoradiography and cultured epidermal sheet explants, and by assessing the LC population in normal mice. The LC density after whole-body irradiation without any cutaneous shielding was not significantly different from that in skin shielded during whole-body irradiation (P > 0.05), indicating that the additional irradiation to the skin did not contribute to a decrease in LC density. In both instances, the LC number gradually decreased in a linear fashion. The results indicate that epidermal LCs continuously leave the epidermis and are continually replaced by circulating precursor cells from the bone marrow at a steady rate. Autoradiographic studies after a pulse injection of 3H-thymidine showed a labelling index of 0.013%, indicating that local mitosis is not an important contributor to the maintenance of the epidermal LC population. Although local X-irradiation resulted in temporary reduction of LC density, epidermal sheet explant culture obtained immediately after local X-irradiation showed no difference in LC density as compared with control unirradiated skin, indicating that the decrease in LC density was not due to significant LC destruction. From these data, we calculated that the half-life of murine LCs in the epidermis is approximately 9 days.     

Plasmacytoid dendritic cells: a new cutaneous dendritic cell subset with distinct role in inflammatory skin diseases

Epidermal dendritic cells found in inflamed skin include Langerhans cells and the recently identified population of inflammatory dendritic epidermal cells. Another subset of dendritic cells in humans is the plasmacytoid dendritic cell in peripheral blood, which is characterized by the production of large amounts of type I interferon (interferon-alpha and interferon-beta) upon viral infection. We hypothesized that plasmacytoid dendritic cells might be involved in anti-viral defense mechanisms of the skin. Here we investigated plasmacytoid dendritic cells, inflammatory dendritic epidermal cells, and Langerhans cells in epidermal single cell suspensions of normal looking skin from healthy volunteers and of lesional skin from patients with different inflammatory skin diseases. Langerhans cells were found in normal and in inflamed skin samples. In normal skin, plasmacytoid dendritic cells and inflammatory dendritic epidermal cells were low or absent. Lesional skin samples from patients with psoriasis vulgaris and contact dermatitis contained relatively high numbers of both inflammatory dendritic epidermal cells and plasmacytoid dendritic cells. In contrast, many inflammatory dendritic epidermal cells but only very few plasmacytoid dendritic cells could be detected in atopic dermatitis lesions. Lupus erythematosus was characterized by high numbers of plasmacytoid dendritic cells but low numbers of inflammatory dendritic epidermal cells. These results demonstrate that in addition to resident Langerhans cells, plasmacytoid dendritic cells and inflammatory dendritic epidermal cells are selectively recruited to the skin lesions depending on the type of skin disease. The lack of plasmacytoid dendritic cells in atopic dermatitis may predispose atopic dermatitis patients to viral infections such as eczema herpeticum, a secondary infection of atopic dermatitis lesions with herpes simplex virus. The composition of dendritic cell subsets may help to clarify the etiology of inflammatory skin diseases and forms the basis for therapeutic intervention with selective microbial molecules such as immunostimulatory CpG oligonucleotides.     

Mechanisms in allergic skin disorders

Background Allergic skin disorders are initiated and maintained through the action and interaction between resident tissue cells, infiltrating leucocytes and mediators. Review The major cellular components of the skin immune system are keratinocytes, dendritic cells such as epidermal Langerhans cells (LCs) and dermal dendritic cells, mast cells and fibroblasts. Following an allergenic stimulus, inflammatory and immunocompetent cells accumulate in the epidermis and dermis, giving rise to specific clinical and histological manifestations. Leucocytes which accumulate in the inflammatory focus attract more leucocytes and stimulate the resident cell population. Primary mediators, released by resident cells upon stimulation, play a vital role in initiating and controlling inflammation. The mediators induce vascular endothelial cells to express adhesion molecules, which mediate migration of leucocytes into the dermis, where chemotactic factors attract them to the inflammatory focus. Activation of trapped T-lymphocytes by antigen-presenting cells (APCs) is followed by the release of inflammatory cytokines. Allergic contact dermatitis (ACD) depends on the activation of specifically sensitised T-cells, and epidermal LCs are the primary APCs. Most contact allergens are small, chemically reactive moleculed and CD4⁺ Th1 type T-cells are the target cells. Atopic dermatitis is a chronic eczematous condition, involving antibody IgE, LCs and dermal dendritic cells as the APCs, with Th2 T-lymphocytes as the target cells. Urticaria comprises a heterogeneous group of conditions, characterised by a wheal and flare reaction. Mast cells are the principal source of mediators involved and histamine is the most important of these.     

Isolation and culture of panning method-enriched Langerhans cells from dispase-dissociated epidermal cells of the mouse

Langerhans cells (LCs) are bone marrow-derived, Ia-positive antigen-presenting cells in the epidermis which constitute 2-4% of the total epidermal cells. We examined the usefulness of a combination of dispase treatment and the panning method for enriching and culturing mouse LCs. Trunk skin was treated with partially purified dispase (Godo Shusei, type II) to separate epidermal sheets and to dissociate epidermal cells. Suspended cells were treated with ascites or culture supernatant containing anti-Ia monoclonal antibody, and LCs were enriched by the Ia-mediated panning method. Per mouse, 3-4 X 10(5) LCs were recovered with greater than 95% purity and greater than 90% viability. Enriched LCs potently stimulated the allogeneic mixed-leukocyte reaction. Ultrastructural observations revealed that enriched LCs contained many vesicles but almost no Birbeck granules. A laminal structure, which was apparently adhesive to the surface of LCs, was observed when ascites were employed as the anti-Ia antibody. These results indicate that a combination of dispase treatment and the Ia-mediated panning method is very useful for isolating high yields of functionally mature murine Langerhans cells with high purity and viability.     

Ultrastructural morphometry of epidermal Langerhans' cells: Introduction of a simple method for a comprehensive quantitative analysis of the cells

Sumary The ultrastructural demonstration of Birbeck granules is the most exact morphological marker for Langerhans' cells (LCs). We have developed a specific ultrastructural morphometric technique that enables quantitative evaluation of the numerical and volume density of LCs as well as quantification of cellular details. Evaluation of normal human skin of the hand and lower buttock showed that the epidermis of the hand contained a significantly higher numerical and volume density of LCs. At the cellular level, these LCs exhibited signs of increased metabolic activity, as reflected by the morphometric data of the nucleus, mitochondria, Golgi area, and Birbeck granules. The present technique thus allows functional characterization of LCs at the morphological level.     

CD24a expression levels discriminate Langerhans cells from dermal dendritic cells in murine skin and lymph nodes

Langerhans cells (LCs) and dermal dendritic cells (dDCs) are the professional antigen-presenting cells of the skin. Recently, their immunogenic versus tolerogenic role has come under re-investigation. LCs are distinguished from dDCs by Langerin (CD207) staining or by detection of Birbeck granules. However, for in vitro experiments it is desirable to have a simple and robust flow cytometric demarcation of both cell types. We show here that CD24a is expressed on LCs but not on dDCs isolated directly from the skin. Moreover, in combination with major histocompatibility complex class II (MHCII), CD24a expression levels distinguish LCs from dDCs in skin-draining lymph nodes after antigen activation and migration. High expression of CD24a correlated strictly with CD207 expression. MHCII(high) cells were unique for skin-draining lymph nodes and were shown to be the only cells carrying antigen after FITC painting of the skin. CD24a expression levels further differentiated LCs and dDCs in the MHCII(high) population. As staining for CD24a does not require fixation of cells, CD24a-stained cells can be used for in vitro experiments to analyze and compare the functional roles and properties of dDCs and LCs.     

In vivo activation of langerhans cells and dendritic epidermal T cells in the elicitation phase of murine contact hypersensitivity

Langerhans cells (LCs) and dendritic epidermal T cells (DETCs) constitute the skin immune system. To demonstrate the kinetics of in vivo activation of murine LCs and DETCs in the elicitation phase of contact hypersensitivity, we measured the cell area positively stained for I-A and gammadeltaT-cell receptor (or Thy-1.2), respectively, under a fluorescence microscope at various time intervals after topical application of dinitrofluorobenzene. The fluorescence-positive area of LCs increased in parallel with that of DETCs at 1 h and 24 h, indicating the biphasic activation of LCs and DETCs. Early activation was hapten-specific and often exhibited close LC-to-DETC apposition. Experiments with in vivo administration of neutralizing anticytokine antibodies revealed that none of interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and interleukin (IL)-1beta were involved in the induction of early activation of LCs and DETCs, while TNF-alpha and IL-1beta mediated late activation of LCs, and IFN-gamma and IL-1beta mediated that of DETCs. Our results indicate that LCs and DETCs are synchronously and biphasically activated in the epidermis during the elicitation phase of contact hypersensitivity and suggest that different mechanisms may control early and late activation.     

Electron microscopic study on Langerhans cells and related cells in lymph nodes of DNCB-sensitive mice

To understand contact hypersensitivity, it is important to know the kinetics of Langerhans cells (LC) and related cells in the lymph node (LN), as well as in the skin. For this purpose, we tried experimentally to induce increased numbers of LCs, Birbeck granule-like structure (BgS)-containing cells, and interdigitating reticulum cells (IDC) in DNCB-sensitive mice and studied them by means of electron microscopy with the following results: (1) cytologically, LC, BgS-containing cells and IDC were closely related; (2) BgS seemed to arise from rough-surfaced endoplasmic reticulum (r-ER), and BgS-containing cells were midway in nature between LC and IDC from the morphological view point. From these findings, it appears that IDC, BgS-containing cells, and LCs were simultaneously involved in the contact hypersensitivity reactions of LNs.