Identification of an equivalent to murine Thy-1+ dendritic epidermal cells in the rat epidermis.

In the murine epidermis, there exist Thy-1+ dendritic epidermal cells (Thy-1+DEC). These cells are Thy-1+, CD45+, CD3+ and asialo GM1+ but CD5-, CD4-, CD8-, or Ia-1-, and express T cell receptor (TCR) gamma delta. Recently, most of these TCR gamma delta of Thy-1 DEC are shown to consist of a V gamma 3-V delta 1 combination. There has been no evidence that the same type of cell population exists in other species except mice. In this study, we investigated the existence of a Thy-1+DEC equivalent in the rat epidermis. The epidermal sheets obtained from rats were stained with various monoclonal antibodies to rat lymphocytes. We developed a monoclonal antibody (1F4) to rat CD3 complex. 1F4 stained thymocytes and peripheral T cells and also immunoprecipitated T cell receptor with CD3 complex. Using 1F4 and a recently developed monoclonal antibody to rat TCR alpha beta, we could identify dendritic CD4-, CD8-, CD5-, CD3+, TCR alpha beta- cells in the rat epidermis. These CD3+, TCR alpha beta- cells are strong candidates as an equivalent to TCR gamma delta + murine Thy-1+ DEC.     

Thy-1+ dendritic cells in murine epidermis are bone marrow-derived

Thy-1+, Ly-5+ dendritic cells have recently been described as a resident cell population in murine epidermis, but their ontogeny and function are unknown. We therefore investigated the origin and turnover of epidermal Thy-1+ cells utilizing chimeric mice. Lethally x-irradiated AKR/J (Thy-1.1+) and AKR/Cum (Thy-1.2+) mice were reconstituted with allogeneic bone marrow cells with or without thymocytes from congenic AKR/Cum or AKR/J mice, respectively. The density of residual indigenous Thy-1.1+ cells in AKR/J chimeras and Thy-1.2+ cells in AKR/Cum chimeras was substantially reduced following x-irradiation, as determined by immunofluorescence staining of epidermal sheets. Epidermal repopulation by allogeneic Thy-1+ dendritic epidermal cells was first observed at 5 weeks in AKR/J chimeras and at 7 weeks in AKR/Cum chimeras and progressed slowly. Repopulation was not enhanced by increasing the number of allogeneic bone marrow cells injected from 2 X 10(7) to 10(8) cells or by the addition of 8 X 10(7) allogeneic thymocytes to the donor inoculate. Epidermal repopulation by allogeneic Thy-1.2+ cells was not seen in AKR/J mice reconstituted with syngeneic bone marrow cells and allogeneic Thy-1.2+ AKR/Cum thymocytes. Taken together, these results indicate that Thy-1+ dendritic epidermal cells are derived from the bone marrow and suggest that they are not related to conventional peripheral T-lymphocytes.     

Thy-1 antigen-bearing dendritic cells populate murine epidermis

Two distinct cell populations, melanocytes and Langerhans cells (LC), have been recognized previously to possess dendritic configuration in normal mammalian epidermis. Employing immunofluorescence microscopy with monoclonal antibodies against Thy-1.2 antigen to identify cells in whole mounts of murine epidermis, we have identified a third dendritic cell population which differs from both LC and melanocytes. Thy-1 antigen-bearing (Thy-1+) epidermal cells are primarily dendritic, although round and angular forms may be found. They are distributed relatively evenly across skin surfaces, although densities vary greatly from site to site and from strain to strain. Densities were highest in ear epidermis from the pigmented strain B10.A (580 cells/mm2), a value approaching that of epidermal LC, and were lowest in ear epidermis from the albino strain BALB/c (5 cells/mm2). Thy-1+ epidermal cells possess neither Ia antigens nor substantial amounts of melanin, and their surface distributions are disparate from those of both LC and mature melanocytes. We propose that at least some of these cells are T lymphocytes whose malignant counterparts account for cutaneous T-cell lymphomas.     

Culture and characterization of murine dendritic Thy-1+ epidermal cells

Although numerous advances have been made in characterizing the phenotype, ontogeny, ultrastructure, and cytochemistry of the murine Thy-1+ dendritic epidermal cell (Thy-1+ EC), elucidation of its functional qualities has been hampered by the difficulty in preparing pure populations of these cells. We therefore sought to obtain expanded, purified populations of Thy-1+ EC using culture techniques. Since Thy-1+ EC are bone marrow-derived, density gradient enriched populations of freshly harvested epidermal cells (FH-EC) were placed in culture under conditions known or suspected to promote mitogenesis among leukocyte subsets. FH-EC prepared from truncal skin of C3H/HeN mice (Thy-1.2+) were cultured at 37 degrees C in 5% CO2 in complete medium (CM) of Eagle's Hanks' amino acid with 10% fetal calf serum, nutrients, and antibiotics at 10(6) FH-EC/well in 24-well culture plates. CM was supplemented with one or more of the following: concanavalin A (Con-A), interleukin-1/epidermal cell-derived thymocyte-activating factor (IL-1/ETAF), IL-2, IL-3, gamma interferon, indomethacin (IM), and anti-Thy-1.2 antibody. Media with appropriate supplements were changed every 2-3 days. Freshly isolated, enriched FH-EC contained 7-20% Thy-1+ EC (defined as brightly fluorescing cells readily distinguishable from weakly fluorescing keratinocytes), which also stained with antibodies directed against asialo GM1, Ly 5.1, and vimentin but did not stain with antibodies to other T cell-, B cell- or macrophage phenotypic markers. Analysis of 10 separate cultures revealed a 3- to 10-fold expansion of nonkeratinocyte Thy-1+ cells after 21 +/- 4 days in culture in CM supplemented with Con-A and IM, and 70-100% of viable cells after expansion were Thy-1+. Phenotypic analysis of expanded cells revealed the emergence in 10 separate cultures of one of two mutually exclusive distinct populations: one Thy-1+, asialo GM1+, L3T4- (natural killer phenotype) and the other Thy-1+, asialo GM1-, L3T4+ (T helper phenotype). Experiments designed to explain the emergence of an L3T4+ population suggest that phenotypic modulation occurred in vitro.     

The murine (C3H/He) epidermal Ia+ dendritic cells (Ia+DECs) and Thy-1+ dendritic cells (Thy-1+DECs) in contact hypersensitivity and aging

The contact sensitivity evaluated by the ear swelling test and the dynamic changes of epidermal Ia+ dendritic cells (Ia+DECs) and Thy-1+ dendritic cells (Thy-1+DECs) were studied in trinitrochlorobenzene (TNCB) sensitized different age group C3H/He mice after challenge. A significant increase of ear swelling was observed between 6 h and 10 days of both 8-10 week (wk) and 40-48 wk groups; the ear swelling indices of 8-10 wk group were significantly higher than those of 40-48 wk group from 18 h to 5 days. A significant decrease of the densities of Ia+DECs from 18 h to 48 h, followed by a gradual increase reaching significant increase of the densities of Ia+DECs from 5 days to 21 days in both 8-10 wk and 40-48 wk groups, was observed; the densities of Thy-1+DECs significantly decreased from 18-48 h, followed by a gradual increase reaching a significant increase from 5 days to 21 days in both 8-10 wk and 40-48 wk groups. In the normal control groups, a significant decline of both Ia+DECs and Thy-1+DECs in the 40-48 wk group was observed. Results suggest that contact allergy may be diminished in aged mice. On the other hand, like Ia+DECs, Thy-1+DECs seem to be involved in the process of contact allergy.     

Thy-1+ dendritic epidermal cells undergo mitosis in vivo

Recently, morphologic evidence that epidermal Langerhans cells (ELC) undergo a mitotic cycle in normal mouse ear skin has been presented. In the present study, using immunohistochemical staining, we examined the mitotic activity of Thy-1-positive dendritic epidermal cells (Thy-1+DEC) in the normal murine epidermis. A small number of Thy-1+DEC showed round, cleaved, paired, and paired dendritic morphologies, which are identical to those occurring during ELC mitosis. We conclude that normal Thy-1+DEC undergo mitosis within the epidermis to maintain their population in the murine epidermis.     

FACS purification of bone marrow-derived epidermal populations in mice: Langerhans cells and Thy-1+ dendritic cells

A method was developed which allows for the separation and purification of Langerhans cells (LC) and Thy-1+ cells (Thy-1+dEC) from mouse epidermis. Epidermal cell (EC) suspensions were subjected to Ficoll separation, and the resulting interface EC were harvested. These EC were then "tagged" with the appropriate monoclonal antibody and sorted into positive and negative populations using the Fluorescence Activated Cell Sorter (FACS). Preparations of viable LC and Thy-1+dEC were obtained with 94-98% and 94-99% purities, respectively.     

Surface densities of murine Ia+ dendritic epidermal cells (Ia+DECs) and Thy-1+ dendritic epidermal cells (Thy-1+DECs) in relationship to aging and ultraviolet B (UVB) radiation

Identification and enumeration of both Ia + dendritic epidermal cells (Ia + DECs) and Thy-1 + dendritic epidermal cells (Thy-1 + DECs) from various parts of the body and non-irradiated and ultraviolet B (UVB) irradiated back skin were examined using epidermal sheets of C3H/He inbred mice of different age groups and indirect immunofluorescent technique. The following results were obtained: [1] There was a significant decline in both Ia + DEC and Thy-1 + DEC density in the mice in the oldest group (48–50 weeks); [2] The densities of Ia + DECs were significantly higher than those of Thy-1 + DECs in comparisons of various parts of the body; [3] At 24 h after 60–120 mJ/cm² UVB irradiation, the Ia + DECs and Thy-1 + DECs decreased significantly in a dose-dependent fashion. The Ia + DECs decreased drastically (p<0.01) while the Thy-1 + DECs decreased mildly (p<0.05). [4] The degree or resistance to UVB differed between Ia + DECs and Thy-1 + DECs in older mice (40–48 weeks). These findings may imply that the decline of the Ia + DECs and Thy-1 + DECs reflects alterations in immune response during aging; As do Ia + DECs, Thy-1 + DECs might also play a role in UVB induced specific unresponsiveness in contact hypersensitivity; each type of Ia + DECs and Thy-1 + DECs follows a distinct biological kinetic pattern after UVB irradiation.     

Thy-1 antigen-bearing dendritic cells in murine epidermis are derived from bone marrow precursors

Thy-1 antigen is expressed on a dendritic subpopulation of cells in murine epidermis. Numbering between 200 and 500/mm2 surface area in abdominal skin, they are distinct from the dendritic Langerhans cells (LCs) and melanocytes. Since immigrant lymphoid cells as well as constitutive cells in various organs have been demonstrated to be Thy-1+, their origin and function are not certain. To assess these issues, two experimental protocols were established. First, grafts of whole skin from AKR mice were placed orthotopically on (AKD2)F1 recipients. Immigration of recipient-derived cells into graft epidermis was assessed histologically by fluorescence microscopy employing monoclonal anti-Thy-1.2 and anti-I-Ad antibodies. Second, bone marrow chimeras were established in AKR recipients after lethal irradiation and reconstitution with cells from (AKD2)F1 donors. In the first protocol, dendritic I-Ad+ LCs of donor origin infiltrated each graft to normal densities within 2 weeks. Thy-1.2+ cells also immigrated into the same grafts, but at much slower rates. In the second protocol, bone marrow-derived Thy-1.2+ cells populated normal skin epidermis slowly over several months, with densities reaching 70/mm2. We conclude that some, if not all, Thy-1+ cells in normal murine epidermis are derived from bone marrow precursors, that their infiltration rates differ substantially from those of LCs, and that those factors which govern immigration rates into adult skin derive from the skin itself rather than from the systemic availability of their precursors. We suggest that the function of Thy-1+ epidermal cells will therefore reside among those usually ascribed to recirculating hematogenous cells, including the possibility that they may down-regulate immunizing signals that emerge from skin.     

小鼠Thy-1阳性树突状表皮细胞密度的两性差别

现已证实在小鼠不同品系下同部位Thy-1阳性树突状表皮细胞(Thy-1⁺dEC)密度存在明显差异.至今尚无有关其两性间差别的报道.     

Sunscreens protect epidermal Langerhans cells and Thy-1+ cells but not local contact sensitization from the effects of ultraviolet light.

This study compares the ability of two commonly used sunscreens--octyl dimethyl para-aminobenzoate (Padimate O) and 2-ethylhexyl-p-methoxycinnamate (2-EHMC)--to protect Langerhans cells (LC), Thy-1+ dendritic epidermal cells (Thy-1+ dEC), and local contact sensitivity (CS) from the effects of ultraviolet (UV) light. Chronic exposure of mice 5 d per week for 4 weeks with an intermediate dose of solar-simulated sunlight from which any UVC had been filtered reduced the LC and Thy-1+ dEC density of murine epidermis. This irradiation procedure was designed to simulate closely the daily exposure of humans to sunlight. This effect on LC and Thy-1+ dEC occurred in both albino and pigmented mice that develop a tan during the irradiation procedure, indicating that a tan does not protect these cells from the effects of UV light. Sunscreen preparations with Padimate O and 2-EHMC, both of which also contained benzophenone-3, as well as Padimate O or 2-EHMC in organic solvent, inhibited UV light from depleting LC from the epidermis of both mouse strains. Padimate O and 2-EHMC in organic solvent were used to ensure that these were the active ingredients in the sunscreen preparations. In contrast to the effects on LC, Padimate O, but not 2-EHMC, protected Thy-1+ dEC from UV exposure in both mouse strains, but neither protected against the development of local immunosuppression using a contact sensitivity model. Thus, even in a mouse strain that is sensitive to UV-induced immunosuppression, local immunosuppression can occur in the presence of normal densities of LC and Thy-1+ dEC.     

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.     

Fluorescence microscopic and flow cytometric analysis of bone marrow-derived cells in human epidermis: a search for the human analogue of the murine dendritic Thy-1+ epidermal cell

Lymphoid cells with an affinity for the epidermis (epidermotropic lymphocytes) have been proposed to play a role in the immune functions of the epidermis. However, antigen-presenting Langerhans cells (LC) and indeterminate cells are presently the only cells in the human epidermis which have been demonstrated to originate in the bone marrow. Recent studies of murine epidermis have identified a population of bone marrow-derived cells which express Thy-1 antigen and which are present in a similar density to, but distinct from, LC. We therefore sought to identify the potential human analogue of the murine Thy-1+ epidermal cell utilizing a battery of antileukocyte reagents in immunohistochemical, flow cytometric, and cell sorting studies. A panel of antibodies failed to detect significant numbers of human Thy-1 antigen-bearing cells, T cells, B cells, monocytes/macrophages (other than LC), and natural killer cells in tissue sections, epidermal sheets, and epidermal cell (EC) suspensions. This was the case using EC suspensions either unfractionated or fractionated on Ficoll-Hypaque to enrich for leukocyte subpopulations. Since the nature of the murine Thy-1+ EC is uncertain, it is possible that antibodies directed against well-defined leukocyte subpopulations may not be of value in the detection of a potential human analogue. We therefore utilized double fluorescence staining with anti-HLe-1, an antibody which identifies all human leukocytes, and anti-HLA-Dr (Dr), which identifies epidermal LC, in order to demonstrate a potential population of HLe-1+ Dr- non-LC, bone marrow-derived cells. The vast majority of HLe-1+ cells were HLA-Dr+ LC; these were present at a density of 608 cells/mm2 in epidermal sheets. A minor population of HLe-1+ cells which did not express HLA-Dr (HLe-1+ Dr-) was observed in tissue sections, epidermal sheets, and EC suspensions. The nondendritic morphology and low density of these HLe-1+ Dr- EC in epidermal sheets (mean density of 4.2 +/- 1.6 cells/mm2) precluded their representing a strict human analogue of the murine Thy-1+ EC, since murine Thy-1+ EC are dendritic and are present in a density similar to that of LC. Purified preparations of the minor HLe-1+ Dr- EC population obtained by electronic cell sorting or panning and examined ultrastructurally were not enriched for any bone marrow-derived cell population. Thus, using currently available markers and sorting technology, we have been unable to identify a human analogue of the murine dendritic Thy-1+ epidermal cell.     

Expression of Thy-1 antigen by murine epidermal cells

We report on the occurrence of a cell population within the murine epidermis which, by both morphologic and surface property criteria, is distinct from all other epidermal cell types known so far. These previously unrecognized cells are evenly distributed within the epidermis, display a primarily dendritic shape, exhibit a lobulated nucleus, contain large amounts of vimentin type intermediate-sized filaments, but lack desmosomes, melanosomes, Merkel cell granules, and Birbeck granules. As opposed to melanocytes, these cells fail to display tyrosinase activity. Surface marker analysis reveals these cells to uniformly express the Thy-1 antigen and to lack I-A and I-E/C antigen specificities. A major portion of these Thy-1-bearing cells are reactive with a monoclonal antibody to the Ly-5 determinant whereas attempts to demonstrate Lyt-1,2,3 antigens consistently yield negative results. These findings strongly suggest that Thy-1+ epidermal cells originate from the bone marrow; however, their precise relationship to distinct members of the hemopoietic differentiation pathway remains to be established.     

Freshly isolated Thy-1+ dendritic epidermal cells express T cell receptor gamma delta-CD3

Within murine epidermis exists a population of Thy-1+ DEC which express membrane Thy-1 antigen, but lack CD8 or CD4 antigen. We examined freshly obtained non-cultured Thy-1+ DEC both by immunofluorescence and by biochemical techniques to identify the protein products of the T cell receptor (TCR) and the associated CD3 complex on these cells. Virtually all of the Thy-1+ DEC are brightly positive in CD3 expression with immunofluorescence using the monoclonal antibody 145-2C11. By immunoprecipitation, using this same antibody and polyclonal anti-TCR-gamma antibody, the only TCR heterodimer detected on the freshly isolated Thy-1+ DEC is the gamma delta heterodimer. These findings suggest that in the phenotype and TCR expression, Thy-1+ DEC are analogous to CD8-, CD4- early fetal thymocytes.     

Thy-1+ dendritic epidermal cells in mice: precursor frequency analysis and cloning of concanavalin A-reactive cells

Bulk cultures of mouse Thy-1+ dendritic epidermal cells (Thy-1+ DEC) have been shown to proliferate in response to concanavalin A (Con A) and IL-2, to secrete IL-2-like growth factors, and to lyse target cells such as YAC-1. Limiting dilution microculture was utilized in order to determine the precursor frequency of Con A-responsive Thy-1+ DEC in suspensions of AKR/J epidermal cells as well as whether these several functional activities all reside within a single Thy-1+ DEC precursor. Precursor frequency analysis of cultures established with limiting numbers of FACS-purified Thy-1+ DEC, irradiated syngeneic splenic filler cells and exogenous IL-2 indicated that approximately 20% of Thy-1+ DEC proliferated in response to Con A. Parallel microcultures in which purified Thy-1+ DEC were plated at a density of 0.5 cells/well were used to establish clones. Twenty clones were characterized phenotypically, and ten of these were also tested for their capacities to proliferate in response to Con A or IL-2, to secrete IL-2-like growth factors, and to exhibit cytotoxicity. All clones were Thy-1+ and L3T4-, but while most were also Lyt-2-, several contained 3%-18% dull Lyt-2+ cells. Functional studies revealed that each clone displayed all of the above functional activities, albeit with substantial quantitative variation. Clones with the highest cytotoxic activity had relatively low responsiveness to Con A or IL-2 and included all clones containing dull Lyt-2+ cells; conversely, clones with the highest proliferative responses had relatively low cytotoxic activity and were all Lyt-2-. This degree of functional and phenotypic heterogeneity among cloned Thy-1+ DEC may reflect their particular states of activation or differentiation; whether it reflects the biologically relevant in vivo activities of these cells must still be determined.