Three-dimensional reconstruction and stereometric analysis of Langerhans cells in mouse epidermis.

In the presented studies stereometric analysis and spatial reconstruction was performed on two Langerhans cell (LC) types. One was free of LC-I and the other contained LC-II Birbeck granules in the perinuclear space. The presented stereometric analysis demonstrated significant differences between the so-distinguished two cell types. Differences were observed not only in the number and distribution of Birbeck's granules but also in the areas of smooth and rough endoplasmic reticulum, in the area of vesicles surrounding Golgi apparatus, in the volume of cisterns of the apparatus, and in the ratio of cell nucleus area to its volume. Differences noted between the two cell types were of quantitative character. They might result from different stages of differentiation of the cells from their precursors in the epidermis or from distinct functional stages of the cells.     

Migration of Langerhans cells into the epidermis of human skin grafted into nude mice.

In a previous study, it was demonstrated that human Langerhans cells (LC) are preserved in human skin grafted onto a nude mouse. Moreover, although it was observed that mouse LC of the host invade skin grafts from allogeneic mouse or rat, they do not penetrate in human skin grafts. In most of the human skin equivalent systems produced in vitro, LC appear to be lost. The present study was designed to investigate whether the mouse LC will repopulate a human skin equivalent. For this purpose, two different systems of skin equivalent have been grafted into the nude mouse. They were composed of human keratinocytes deposited on dead human dermis, or on lattice composed of human fibroblasts embedded in type I collagen. At different times after grafting, the presence of LC in the transplants was assayed either by indirect immunofluorescence or by electron microscopy. Indirect immunofluorescence was performed on frozen sections or on epidermal sheets with anti-Ia, anti-HLA-DR, or OKT6 antibodies. It was observed that, at 2 months after grafting, Ia(+) HLA-DR(-) OKT6(-) cells are present in grafted human epidermis. Moreover, LC with typical Birbeck granules are also detected by electron microscopy. It could be concluded, from this study, that mouse LC can repopulate human epidermis devoid of human LC.     

Langerhans cells in human warts

Seventy-six warts (15 plantar, 38 hand, 16 miscellaneous and seven anogenital lesions) taken from 55 patients, were studied by indirect immunofluorescence with monoclonal antibodies specific for T-cell subsets, Langerhans cells (LC) and HLA-DR antigen. The results were related to the presence of viral antigen. Approximately 80% of the lesions showed an infiltrate. Only 19 lesions contained helper/inducer or suppressor/cytotoxic T cells. The distribution of LC was abnormal in 65% of biopsies which contained LC in the dermis, and 29% were devoid of LC in the epidermis. Many lesions had reduced numbers of LC in the epidermis. The disappearance of LC from the epidermis was related to the presence of viral antigen, but not to the presence of particular T-cell subsets. Infiltrating cells were sometimes HLA-DR-positive, whereas basal cells did not express HLA-DR antigen, irrespective of the density of the infiltrate.     

Ultrastructural changes in epidermal Langerhans cells and melanocytes in response to ultraviolet irradiation, in Australians of Aboriginal and Celtic descent

The effects of exposure to small doses of artificial ultraviolet radiation (UVR) on the ultrastructure of epidermal Langerhans cells (LC) and melanocytes were studied in two groups of Australian subjects, one of Aboriginal and the other of Celtic descent. UV exposure induced an apparent depletion of LC in the epidermis of both groups. However, LC depletion in the Aboriginal subjects was associated with apoptosis, whereas organelle and membrane disruption in the LC of Celtic subjects suggested a reduction by direct cellular damage. LC in Aboriginal epidermis tended to become relocated at more superficial levels following UV exposure, and their Birbeck granules became more numerous. LC in Celtic epidermis appeared to become relocated in a basal location and contained fewer Birbeck granules. The central lamina of the Birbeck granules in Aboriginal LC, which was more electron-dense than that in Celtic subjects prior to UV treatment, was temporarily lost following treatment, while the ultrastructure of Birbeck granules in Celtic LC was unchanged. LC and 'indeterminate cells' in intimate association with lymphocyte-like cells occurred in the basal layer of Celtic epidermis 5 days after exposure. These complexes were not observed in Aboriginal epidermis although isolated lymphocyte-like cells were observed in the same location. Melanocytes in Aboriginal epidermis contained greater numbers of melanosomes than those in Celtic epidermis throughout the experiment. Inactive epidermal melanocytes in Celtic subjects initially responded to UV exposure with a slight increase in melanosome content followed by a substantial further increase, whereas active melanocytes in the Aboriginal subjects showed the opposite response. The implications of the different responses of LC and melanocytes in the two groups, in relation to immunological function of the epidermis and the marked racial difference in the incidence of skin cancer, are discussed. Cancer of the skin, particularly basal and squamous cell carcinoma, occurs primarily in people with fair skin who burn easily following exposure to ultraviolet radiation (UVR). In contrast, the incidence of skin cancer in inherently dark-skinned people is low. Melanin is synthesized by melanocytes in response to UVR and is thought to protect epidermal cells against damage to their genetic material by absorbing UVR and thereby reducing its penetration into the skin. Thus darkly pigmented skin is more resistant to the effects of UVR.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cellular and subcellular distribution of 2,4-dinitrophenyl groups in mouse epidermis and regional lymph nodes after epicutaneous application of 2,4-dinitrofluorobenzene

Summary The cellular and subcellular distribution of 2,4-dinitrophenyl (DNP) groups in the epidermis and regional lymph nodes of the mouse was investigated after epicutaneous application of 2,4-dinitrofluorobenzene (DNFB) to sensitized and non-sensitized mice. The peroxidase-antiperoxidase method and the immunogold technique were used to visualize the DNP groups at both light and electron microscopic levels. The highest intensity of immunolabelling was found on tonofilaments of keratinocytes present in the upper layers of the epidermis. On the other hand, in vitro experiments showed that DNFB has the capacity to bind keratin which, together with immunocytochemistry, suggests that this molecule may be one of the skin protein carriers for DNFB. In addition, intense immunostaining for DNP was observed in the Golgi area of some epidermal Langerhans cells. Cells immunoreactive to DNP were also observed in the marginal sinus of cervical lymph nodes 6, 12 and 24 h after challenge. Immunoelectron microscopy revealed immunoreactive DNP groups in phagosomes of Langerhans cells at this site. The present findings support the hypothesis that the hapten DNFB penetrates passively into the cytoplasm of Langerhans cells, concentrates in the Golgi area and, during the migration of Langerhans cells to the lymph nodes, it is probably processed in the lysosomes before its presentation to T lymphocytes.     

An immunohistochemical and immunoelectron microscopic study of the ontogeny of rat Langerhans cell lineage with anti-macrophage and anti-Ia monoclonal antibodies

An immunohistochemical study with anti-macrophage and anti-Ia monoclonal antibodies was performed to clarify the relationship between Langerhans cells (LC) and indeterminate cells (IC) in rat epidermis both in adulthood and in the fetal stage. On immunoelectron microscopy, a mouse anti-rat macrophage monoclonal antibody, TRPM-1, recently produced by us, reacted with IC and some LC in adult rat skin. Ontogenic study revealed that TRPM-1-positive cells first appeared in the epidermis of fetal rat heads on Day 17 of gestation and then spread caudally along the anterior-posterior axis. On Day 20 of gestation, when the distribution of the TRPM-1-positive cells over body surface became even, Ia-positive cells appeared in the epidermis and began to increase in number. Ia-positive cells with Birbeck granules were found on Day 21 of gestation. These results indicate that. TRPM-1-positive IC matured into Ia-expressing LC after being exposed to microenvironmental change during the perinatal period. The number of Ia-positive cells exceeded that of TRPM-1-positive cells at around 5 d after birth. Afterwards, there were more dendritic Ia-positive cells found in the interfollicular areas than TRPM-1-positive ones. However, local concentrations of the TRPM-1-positive IC in the follicular infundibula were frequently found in the fetal stage and occasionally in adulthood. These TRPM-1-positive cells in the follicular infundibula were thought to be a precursor pool in the epidermis for LC.     

Distributional changes of Langerhans cells in human skin during irritant contact dermatitis

We used light and electron microscopic immunocytochemistry to study distributional changes in the human Langerhans cell (LC) system during the first 14 days of a mild irritancy caused by sodium lauryl sulphate (SLS). A marked initial decrease in epidermal LC was noted possibly resulting from migration from the epidermis to the dermis and from irreversible cell damage. Several studies have previously found an unchanged number of LC in SLS-induced contact irritant dermatitis, but these studies may not have taken into account the fact that SLS is effectively absorbed from the test chamber. Unless certain precautions are taken the SLS concentration rapidly falls to topical levels that have no effect on the LC system. Simultaneously with the decrease in the epidermis we observed an increase in dermal CD1a+ cells, confirming an often reported finding. There is, however, no consensus as to the identity of these cells, and several authors have reported that such cells lack LC granules and thus these cells have often been classed as indeterminate cells. We found that, during irritant contact dermatitis, provided an adequate number of sections were scrutinized in the electron microscope, all dermal CD1+ cells contained Birbeck granules.     

Comparative epidermal Langerhans cell migration studies in epidermal and epidermal/dermal equivalent grafts.

Immigration of Langerhans cell precursors from the peripheral blood to the skin was studied in human grafts placed on severe combined immunodeficient (SCID) mice. Monocyte fractions of human blood were injected intraperitoneally to SCID bearing either reconstituted (Langerhans cell free) epidermal sheets (E) or living skin equivalents (E/D) consisting of both epidermis and dermis. A range of immunocytochemical and ultrastructural markers was employed to monitor the colonization of the grafts, i.e., CD1a/c, Birbeck granules. In situ hybridization with probes against Alu sequences of human DNA were employed together with immunostaining for MHC class I mouse and human antigens to document graft survival. Although unequivocal LC were detected within E grafts, including both human (CD1a positive) and murine (NLDC-145 positive), no migration was achieved in the E/D situations.     

Increased densities of Langerhans cells in the epidermis of skin tumors

Using OKT6 monoclonal antibody (MCA) and β-specific anti-S-100 protein (S-100β) MCA, the numbers of Langerhans cells (LC) in the epidermis overlying squamous cell carcinoma (SCC), basal cell epithelioma (BCE), dermatofibroma (DF), and in the lesional epidermis of seborrheic keratosis (SK) were investigated. The numbers of LC were significantly increased in the epidermis overlying SCC and in SK. Moreover, significantly more LC were observed in the epidermis overlying SCC than in SK. The increased density of LC in the epidermis overlying SCC may suggest participation and activation of the mononuclear phagocyte system in this neoplasm. In 8 cases of SCC, the subpopulations of infiltrating lymphocytes around the tumor cell nests were analyzed. The ratio of Leu 3a-positive cells/Leu 2a-positive cells ranged from 0.63 to 1.99. This diversity may reflect complicated immunological interaction between the tumors and the hosts.     

Accessory and adhesion molecules expressed on murine epidermal Langerhans cells and the modulation by cytokines.

Langerhans cells (LC) are MHC class II (Ia)-positive dendritic cells that act as an antigen-presenting cells for T cell-dependent immune responses. LC originate from cells in bone marrow and migrate into the epidermis through blood vessels. LC also migrate from the epidermis to regional lymph nodes after antigen stimulation where they present antigens to T cells. These are the essential features of LC. The morphological and functional properties of LC are modulated by external stimuli or various cytokines. In this review we focus on the accessory and adhesion molecules on LC and describe how these molecules are modulated by cytokines. We also describe the molecules participating in the migration of LC into and from the epidermis. Moreover, we introduce our data obtained from purified murine epidermal LC and from the transgenic mice overexpressing several cytokines in the epidermis.     

Quantitative studies of the fate of epidermal Langerhans cells after X-irradiation of guinea-pig and mouse footpad skin

Langerhans cell numbers, morphology and distribution were observed in cross sections of footpad epidermis at intervals from 1 to 28 days after exposure of the hind feet of CBA/H mice or albino guinea-pigs to a single absorbed dose of 20 Gy (2000 rad) of X-rays. In mice, the number of Langerhans cells reactive with anti-macrophage F4/80 monoclonal antibody steadily declined by approximately 85% within 10 days after irradiation, consistent with previous studies, in which Langerhans cells were identified in epidermal sheets by ATPase activity or presence of Birbeck granules. Remaining Langerhans cells were exceptionally dendritic. Very few Birbeck granule-containing cells were found in murine popliteal lymph nodes before or after irradiation but damaged cells were present in superficial strata of irradiated epidermis. The morphology and number of epidermal F4/80-positive cells approached normal by 15 days after irradiation. In guinea-pigs, gradual suprabasal movement and loss of rounded, ATPase-positive Langerhans cells from the epidermis were detectable from 5 to 20 days after irradiation but the magnitude of the cell loss and redistribution was partially obscured by the simultaneous appearance of clusters of replacement Langerhans cells in the basal layer and by keratinocyte hyperplasia.     

Epidermal Langerhans cells--a cycling cell population

The limited number of Langerhans cells (LC) in human epidermis and the resultant technical difficulties have left open the question of LC kinetics. In the present study using flow cytometry (FCM) we have applied 3 methods to estimate LC-DNA distribution: (1) FCM-DNA measurement on highly enriched LC suspensions, (2) FCM-correlated analysis of DNA and OKT-6(+) cells in total epidermal cell suspensions, (3) LC-enriched suspensions (70-90%) were FACS (fluorescence-activated cell sorter) sorted on microscopic slides, and stained with the Feulgen technique, and DNA was measured densitometrically. In the latter method, contaminating keratinocytes were counterlabeled with antikeratin serum to eliminate them from LC-DNA estimation. All 3 in vitro analyses clearly showed that human LC are a cycling cell population in the epidermis. The number of LC in S (1.3-3.3%) and G2/M (1.0-2.5%) phase compares with those found for keratinocytes. Assuming that this percentage of keratinocytes in S and G2/M phases is sufficient to maintain the structural integrity of the epidermis, it was suggested that LC may represent a stable, self-reproducing cell population in normal epidermis.     

Congenital self-healing reticulohistiocytosis. Report of the seventh case with histochemical and ultrastructural studies

A new case of congenital self-healing reticulohistiocytosis was studied with S-100 antibody and electron microscopy. Many tumor cells were S-100-positive and contained Langerhans cell (LC) granules and concentrically laminated dense bodies. Octopus-like bodies and wormlike bodies were also found. The formation of LC granules at the cytomembrane was frequently found, not only between the cytomembrane and its own villi, but also between the cytomembranes of adjoining cells. These findings strongly support the cytomembrane origin of LC granules in general.     

Pagetoid Self-Healing Langerhans Cell Histiocytosis in an Infant

We report Langerhans cell (LC) histiocytosis in a male infant who developed numerous papular lesions on the trunk and posterior scalp soon after birth and spontaneously recovered from the disease within 7 months. Histologically S-100-positive cells were detected in the epidermis and papillary dermis, in some lesions mostly in the epidermis. Tumor cells in the epidermis were either clustered, forming nests, or scattered singly in pagetoid fashion. Electron microscopy confirmed the presence of Birbeck granules in these cells. They exhibited many interesting features usually not found in normal LCs, including mitosis, frequent apoptosis, Birbeck granules invaginated in the nucleus, autophagocytosis of Birbeck granules, and active ingestion of extracellular material through Birbeck granules attached to cell membranes. It is suggested that either a strong epidermotropism of tumor cells or a proliferation of the resident LCs of the epidermis is responsible for this intraepidermal growth pattern. Cellular necrosis through very active apoptosis and the superficial nature of the growth might have contributed to the self-healing course in this patient.     

Limited reliability of E-cadherin as a specific marker for in vitro-generated Langerhans cells

Langerhans cells (LC) are a unique population of dendritic cells (DC) found in the epidermis where they can be identified by the expression of CD1a, E-cadherin and cytoplasmic Birbeck granules (BG) as their hallmark. Over the past years many techniques have been described to generate LC in vitro from either monocytes or CD34⁺ hematopoietic cell progenitors. Antibodies against Lag and Langerin (two epitopes associated with BG) and E-cadherin (a Ca²⁺-dependent homophilic adhesion molecule) have been used to detect in vitro-generated LC. In this study we investigated whether the expression of E-cadherin on in vitro-generated CD1a⁺ from either CD34⁺ cells or monocytes is able to discriminate LC from other DC. Our results demonstrate that E-cadherin alone is not a reliable marker to specifically identify in vitro-generated LC.     

Self‐renewal capacity of human epidermal Langerhans cells: observations made on a composite tissue allograft

Abstract: Epidermal Langerhans cells (LC) are dendritic, antigen‐presenting cells residing within mammalian epidermis and mucosal epithelia. When massively depleted, they are replaced by cells of bone‐marrow origin. However, their renewal within normal skin under steady‐state conditions is not precisely known. We observed that epidermal LC within a human hand allograft remain stable in the long term (10 years) and are not replaced by cells of recipient’s origin; furthermore, we observed a Langerhans cell in mitosis within the epidermis 8 years postgraft. These results show that under almost physiological conditions, human LC renew in the epidermis by local mitoses of preexisting cells.     

Ultrastructural similarities between epidermal Langerhans cell Birbeck granules and the surface-connected canalicular system of EDTA-treated human blood platelets

Birbeck granules characterize under the electron microscope epidermal Langerhans cells. These distinctive pentalaminar organelles are indeed not detectable in the possible precursors of human Langerhans cells and tend to disappear in cultured human Langerhans cells. The mechanisms that lead to the appearance of Birbeck granules in epidermal Langerhans cells and to their later disappearance still remain unknown. In the present study we show that the more or less dilated elements of the surface-connected canalicular system of human blood platelets collapse after EDTA treatment. Made up of two parallel limiting membrane and central irregular striated density, these elements show great ultrastructural similarities with the Birbeck granules of human epidermal Langerhans cells. These platelet morphologic changes i) are directly dependent on the EDTA-induced dissociation of the glycoprotein GP IIb-IIIa, the platelet-specific calcium-dependent heterodimer complex, member of the beta 3 integrin subfamily (alpha IIb beta 3) and ii) apparently result from a cross-linking of the dissociated glycoproteins. These findings lead us to propose that in the same manner cells of the Langerhans lineage, on reaching the epidermis, will find themselves in contact with an epidermal specific ligand. Interactions between this epidermal ligand and Langerhans cell receptors could then induce, all along the circuit taken by the ligand-receptor complexes, morphologic modifications, i.e., appearance of structures of Birbeck granule type.     

The semi-quantitative distribution of T4 and T6 surface antigens on human Langerhans cells

We have used indirect immunogold electron microscopy to compare the respective density of cell membrane determinants revealed by OKT6 and OKT4 monoclonal antibodies on normal human Langerhans cells (LC): 12.9 +/- 3.5 gold granules were noted per cell section on OKT4-positive LC whereas 236.8 +/- 23.5 granules were counted per cell section on OKT6-reactive cells. These results confirm that human LC react with OKT4 antibody and they demonstrate a marked quantitative difference on LC surface between the antigenic determinants recognized by OKT6 and OKT4 antibodies.     

Migration of Langerhans cells in an in vitro organ culture system: IL-6 and TNF-alpha are partially responsible for migration into the epidermis

Although it is well established that epidermal Langerhans cells (LC) originate from bone marrow, little is known about the mechanism of this migration into the epidermis from bone marrow. In order to clarify the mechanism of this migration, we constructed an in vitro model. LC were depleted by daily topical application of clobetazole propionate (CP) solution onto the ear of Balb/c mice. Seven days later, ear skin was cut off, separated and co-cultured dermal-side-up with syngeneic (Balb/c), semisyngeneic ((C3H x Balb/c)F1), or allogeneic (C3H) epidermal cells (EC) for 3 days. We found (1) that a marked migration of donor LC into the recipient epidermis was observed in the LC-depleted skin, (2) that only syngeneic LC actively migrated into the recipient epidermis; however, the migration of semisyngeneic and allogeneic LC was detected at very low levels, (3) that the migratory capacity of donor LC was directly proved by a biolabeling technique using donor EC labeled with PKH-26, and (4) that anti-IL-6 and anti-TNF-alpha antibodies inhibited the migration of donor LC into the recipient epidermis. These data demonstrate that the resident LC have the potential to traffic through the dermis into the epidermis in a highly syngeneic-specific fashion, and that IL-6 and TNF-alpha are partially responsible for promoting this migration.