Coated vesicle isolation by immunoadsorption on Staphylococcus aureus cells

Porcine brain coated vesicles were isolated from crude fractions of tissue homogenates by affinity separation using anticlathrin-coated STaphylococcus aureus (Staph A) cells as a solid-phase immunoadsorbent. The specificity of the immunoadsorption was monitored by SDS PAGE analysis and by competitive ELISA assays. SDS PAGE of the material immunoadsorbed from a fraction of porcine bran smooth microsomes showed a selective enrichment in a 180,000 mol wt protein. In an ELISA assay, this protein competed effectively--in binding anticlathrin--with clathrin extracted from a coated vesicle preparation. When the immunoadsorbed fraction was examined by electron microscopy, coated vesicles and vesicle-free cages were found forming a quasicontinuous monolayer on the surface of the Staph A cells. Other particles were not adsorbed, and the controls were free of either clathrin cages or coated vesicles. Upon extensive dialysis (against MES buffer, pH 6.5), similar cages appeared on the surface of anticlathrin-coated Staph A cells reacted with extracted clathrin. This study demonstrates that anticlathrin-coated Staph A cells can be used for the isolation and purification of a homogeneous population of coated vesicles. In addition, the ability of extracted clathrin to bind and to polymerize onto the Staph A cells raises the possibility of using this technique to further explore the conditions required for cage and/or vesicle reconstitution.     

Targeting epidermal Langerhans cells by epidermal powder immunization

Immune reactions to foreign or self-antigens lead to protective immunity and, sometimes, immune disorders such as allergies and autoimmune diseases. Antigen presenting cells (APC) including epidermal Langerhans cells (LCs) play an important role in the course and outcome of the immune reactions. Epidermal powder immunization (EPI) is a technology that offers a tool to manipulate the LCs and the potential to harness the immune reactions towards prevention and treatment of infectious diseases and immune disorders.     

Formation of Langerhans granules seems linked to membrane ATPase activity of epidermal Langerhans cells

The epidermis contains a population of dendritic cells, Langerhans cells (LC), derived from cells originating from bone marrow, bearing receptors for the Fc fragment of IgG and for the C3 fraction of complement and expressing at their surface Ia antigens of the major histocompatibility system. These cells with multiple immunological functions are capable of presenting antigens to immunocompetent cells. The labeling of LC through revelation of their membranous ATPase activity constitutes one of the best available techniques for their visualization. Moreover, the presence of this ATPase activity appears to be a prerequisite for the induction of contact sensitivity, since in the absence of such activity, the epicutaneous application of a hapten induces a state of immunological tolerance. Applying, at a sensitizing dose, 2,4-dinitrofluorobenzene (DNFB) on an untreated guinea-pig skin surface results in a momentary drop in the number of ATPase positive LC in the application zone. Using an improved technique for ATPase labeling (Hanau et coll. submitted for publication, 1985)--which allows one to extend the study from optical to electron microscopy--we observed by electron microscopy the formation, within the LC, of numerous Langerhans granules, concurrent with the loss of ATPase membranous activity. These granules, first described by Birbeck et coll. and specific to LC in the epidermis, have a complex structure. On a section, they may display either a linear aspect (rod-like)--with sometimes a vesicular portion at one extremity (racket-like)--or a circular shape. Whatever their shape, they always show a central striation, which gives them a zipper-like appearance.(ABSTRACT TRUNCATED AT 250 WORDS)     

The binding of epidermolytic toxin from Staphylococcus aureus to mouse epidermal tissue

Summary Fluorescein-labelled epidermolytic toxin (FTC-toxin) ofStaphylococcus aureus and ferritin—toxin conjugate have been prepared and purified. FTC-toxin bound selectively to cryostat and resin-impregnated sections of neonatal mouse skin. Binding was localized at the keratohyalin granules and in the stratum corneum. In an epidermal cell (granular, spinous and basal) preparation, only keratohyalin granules of the granular cells bound FTC-toxin. Ferritin—toxin conjugate bound to skin sections at the same two sites as FTC-toxin and was competitive with the binding of free toxin. Keratohyalin granules in unstained sections had a novel patched appearance under the electron microscope, and the ferritin—toxin conjugate bound preferentially to the electron-lucent areas. In the stratum corneum it was shown by quantitative estimation that the target density decreased as the surface of the tissue was approached.     

Specificity of suppressor cells of the delayed hypersensitivity type for Staphylococcus studied by immunoadsorption

The immunological specificity of T-suppressors obtained from mice after intravenous immunization with corpuscular antigen was shown. The splenocytes of such a mice suppressed DH to staphylococcal antigens, but not to sheep red blood cells. The suppressor cells under study were specifically adhesive to staphylococci.     

Extracellular products of Staphylococcus aureus reversibly inhibit the terminal differentiation of cultured mouse epidermal cells

The effect of extracellular products from Staphylococcus aureus on the differentiation of mouse epidermal cells was studied using an in vitro cell culture system. The extracellular products from a clinical strain of S. aureus isolated from human skin lesions reversibly inhibited the Ca++-induced terminal differentiation of epidermal cells, as determined by their morphology and the extent of cornified envelope formation. This suggests that a similar modification of cell differentiation is involved in the pathogenesis of S. aureus-induced skin disease.     

A developmental study of murine epidermal Langerhans cells

Prospective skin prior to invasion by neural crest cells was dissected from 10.5-day mouse embryos and cultivated in chick embryo hosts. The graft tissue was prepared for the demonstration of both mouse and chick cells, pigment cells, and Langerhans cells. Chick cells were not found in the graft mouse epidermis; however, ATPase-positive and osmium iodide-positive cells were present. Electron microscopic examination revealed that, in younger grafts, only indeterminate cells could be found among the keratinocytes. In older grafts, both indeterminate cells and Langerhans cells with granules were seen. The evidence affirms that epidermal Langerhans cells are not related to pigment cells.Based on the developmental nature of Birbeck (Langerhans) granules from the cytomembrane, it is proposed that the granule no longer be considered as specific to and characteristic of epidermal Langerhans cells. Rather, Langerhans cells should be defined as ATPase-positive, desmosome-free cells within stratified squamous, potentially keratinizing, epithelia. Thus epidermal, ATPase-positive indeterminate cells and such cells with Birbeck granules both should be considered as components of the Langerhans cell series.Normal chick skin does not show ATPase-positive cells. However, when 10.5-day mouse embryo ectoderm was inserted under the ectoderm of chick embryos, the resulting chimeric epidermis possessed ATPase-positive cells. It is proposed that epidermal Langerhans cells are of ectodermal origin.     

乳杆菌抑制金黄色葡萄球菌对HeLa细胞的粘附

对弯曲乳杆菌Lactobacillus crispatus T79-3和T90-1、詹氏乳杆菌Lactobacillus jensenii T118-3和T231-1四株乳杆菌对金黄色葡萄球菌生长的抑制效果以及抑菌成分进行了分析,比较乳杆菌排除、竞争、置换3种不同作用方式对金黄色葡萄球菌粘附HeLa细胞的抑制作用。结果表明4株乳杆菌皆能抑制金黄色葡萄球菌的生长及其粘附HeLa细胞的能力,分析发现4株乳杆菌发挥抑制作用的主要成分是有机酸,同时比较分析乳杆菌3种不同作用方式发现它们对金黄色葡萄球菌粘附HeLa细胞的抑制效果不同,其中,排除作用方式效果最好。另外,乳杆菌对金黄色葡萄球菌粘附HeLa细胞的抑制作用具有浓度依赖性,随着乳杆菌浓度增大,抑制作用增强并逐渐达到饱和。4株乳杆菌中,T79-3粘附能力最强,对金黄色葡萄球菌的抑制作用最强,排除作用方式抑制金黄色葡萄球菌粘附HeLa细胞作用效果较好,提示乳杆菌T79-3有可能作为益生菌防治妇女泌尿生殖道感染。     

Interferon induced in mouse spleen cells by Staphylococcus aureus

Interferon was produced in suspensions of mouse spleen cells treated with Staphylococcus aureus preparations (killed bacteria, culture supernatants, or purified enterotoxin) under a variety of cell culture conditions. The lysate of S. aureus was found to induce high levels of interferon (10^3.1 to 10^4.3 RU/ml) within 72 hr. The crude interferon was concentrated and partially purified by either ammonium sulfate precipitation or adsorption to silicic acid and elution by ethylene glycol-containing buffer. Sequential precipitation with 50 to 80% saturated ammonium sulfate resulted in a three- to seven-fold purification with 60% recovery of activity. Adsorption to silicic acid resulted in a 25- to 80-fold purification with 77% recovery. This material was further analyzed by gel filtration. The antiviral activity induced by S. aureus-treated spleen cells was characterized as due to interferon. Furthermore, the inhibitor was acidlabile and not neutralizable by antiserum against NDV-induced L-cell interferon, thus exhibiting properties of immune (γ) interferon. The partially purified interferon was used to prepare an antiserum in rabbits. This antiserum was able to neutralize mouse interferon induced by several T-cell mitogens, by antigens, and by mixed lymphocyte cultures, while remaining inactive against interferons induced in vitro by viruses or in vivo by Brucella abortus.     

Latent infection of epidermal Langerhans cells in HIV-positive individuals

Epidermal cell suspension otained fron 3 symptom-free HIV-positive individuals were cultured and marked with monoclonal antibodies for the HIV proteins p15, p24 and gp120 in the alkaline phosphatase anti-alkaline phosphatase staining technique. For 2 individuals, cells were positive after 3 days in culture, and for the third, after 4 days. Supernatant from one of the cultures infected allogeneic peripheral blood mononuclear cells. We conclude that epidermal Langerhans cells from symptom-free HIV-positive individuals are latent-infected and are able to produce and release HIV.     

Catecholamines inhibit the antigen-presenting capability of epidermal Langerhans cells

The sympathetic nervous system modulates immune function at a number of levels. Within the epidermis, APCs (Langerhans cells (LC)) are frequently anatomically associated with peripheral nerves. Furthermore, some neuropeptides have been shown to regulate LC Ag-presenting function. We explored the expression of adrenergic receptors (AR) in murine LC and assessed their functional role on Ag presentation and modulation of cutaneous immune responses. Both purified LC and the LC-like cell lines XS52-4D and XS106 expressed mRNA for the ARs alpha(1A) and beta(2). XS106 cells and purified LC also expressed beta(1)-AR mRNA. Treatment of murine epidermal cell preparations with epinephrine (EPI) or norepinephrine inhibited Ag presentation in vitro. Furthermore, pretreatment of epidermal cells with EPI or norepinephrine in vitro suppressed the ability of these cells to present Ag for elicitation of delayed-type hypersensitivity in previously immunized mice. This effect was blocked by use of the beta(2)-adrenergic antagonist ICI 118,551 but not by the alpha-antagonist phentolamine. Local intradermal injection of EPI inhibited the induction of contact hypersensitivity to epicutaneously administered haptens. Surprisingly, injection of EPI at a distant site also suppressed induction of contact hypersensitivity. Thus, catecholamines may have both local and systemic effects. We conclude that specific ARs are expressed on LC and that signaling through these receptors can decrease epidermal immune reactions.     

Mediated exodus of L-dopa from human epidermal Langerhans cells

Summary. L-3,4-dihydroxyphenylalanine (L-dopa) is not metabolized within human epidermal Langerhans cells (LC); yet they can take up substantial amounts of this amino acid which subsequently can be released into the extracellular space. We recently reported that human epidermal energy metabolism is predominantly anaerobic and that the influx mechanism is a unidirectional L-dopa/proton counter-transport system and now we describe conditions for the mediated transport of L-dopa out of the LC. It is demonstrated that certain amino acids and one dipeptide can effectively trigger the efflux of L-dopa taken up by the LC.Thus, -methyl-dopa (-m-dopa), D-dopa and the dipeptide, met–ala at the outside of the plasma membrane stimulated the efflux of L-dopa from L-dopa loaded LC. Similar effects were achieved by a variety of other amino acids in the extracellular fluid while some other amino acids were inactive. The time required for 50% D-methionine-induced exodus of L-dopa from L-dopa loaded LC was in the range of 5–7min and a complete exodus of L-dopa was attained at about 20min of incubation. This dislocation of L-dopa to the extracellular fluid is interpreted as an expression of trans-stimulation. In the case of -m-dopa, D-dopa and met–ala, which admittedly were not able to penetrate the plasma membrane of LC, the concept of trans-stimulation was given a new purport, since none of them were able to participate in an exchange reaction. Finally, it could be concluded that L-dopa escaped by a route different from the one responsible for L-dopa uptake in LC.Thus, while the influx of L-dopa supports extrusion of protons deriving from anaerobic glycolysis in the LC, L-dopa efflux can provide the cells with useful amino acids in an energy-saving way, altogether a remarkable biological process. From this follows that L-dopa has a biological function of its own, besides being a precursor in the catecholamine and pigment syntheses.     

CD34+ -derived Langerhans cell-like cells are different from epidermal Langerhans cells in their response to thymic stromal lymphopoietin

Thymic stromal lymphopoietin (TSLP) endows human blood‐derived CD11c⁺ dendritic cells (DCs) and Langerhans cells (LCs) obtained from human epidermis with the capacity to induce pro‐allergic T cells. In this study, we investigated the effect of TSLP on umbilical cord blood CD34⁺‐derived LC‐like cells. These cells are often used as model cells for LCs obtained from epidermis. Under the influence of TSLP, both cell types differed in several ways. As defined by CD83, CD80 and CD86, TSLP did not increase maturation of LC‐like cells when compared with freshly isolated LCs and epidermal émigrés. Differences were also found in the production of chemokine (C‐C motif) ligand (CCL)17. LCs made this chemokine only when primed by TSLP and further stimulated by CD40 ligation. In contrast, LC‐like cells released CCL17 in response to CD40 ligation, irrespective of a prior treatment with TSLP. Moreover, the CCL17 levels secreted by LC‐like cells were at least five times higher than those from migratory LCs. After maturation with a cytokine cocktail consisting of tumour necrosis factor‐α, interleukin (IL)‐1β, IL‐6 and prostaglandin (PG)E₂ LC‐like cells released IL‐12p70 in response to CD40 ligation. Most importantly and in contrast to LC, TSLP‐treated LC‐like cells did not induce a pro‐allergic cytokine pattern in helper T cells. Due to their different cytokine secretion and the different cytokine production they induce in naïve T cells, we conclude that one has to be cautious to take LC‐like cells as a paradigm for ‘real’ LCs from the epidermis.     

Effects of physicochemical agents on murine epidermal Langerhans cells and Thy-1-positive dendritic epidermal cells

The possibility that Thy-1-positive dendritic epidermal cells (Thy-1+DEC) may contribute to the immunologic functions of murine epidermal cells (EC) prompted us to simultaneously assess the effects of certain immunomodulating physicochemical agents on both Thy-1+DEC and Ia-bearing Langerhans cells (LC). C3H/He mice received one of the following treatment modalities: UV-B irradiation (four consecutive days); psoralen plus UV-A (PUVA; three times a week for three consecutive weeks); topically and systemically applied glucocorticosteroids (GCS). Beginning 2 days after the last treatment, animals were sacrificed and the structure and surface marker expression of Ia+EC and Thy-1+DEC were assessed by immunohistologic means on epidermal sheet preparations from ear skin by using appropriate monoclonal antibodies. Whereas low-dose UV-B irradiation (4 X 100 or 200 J/m2) had little, if any, effect on either Ia+EC or Thy-1+DEC, high-dose UV-B (4 X 700 or 1000 J/m2) or PUVA treatment led to an almost complete disappearance of both surface characteristics. Immunoelectron microscopic studies revealed that in the case of LC, high-dose UV-B or PUVA treatment results in the disappearance of their anti-Ia reactivity but leaves their ultrastructural morphology intact. In sharp contrast, Thy-1+DEC escape ultrastructural detection after PUVA treatment and are greatly reduced in number after high-dose UV-B. Ia+EC continuously reappeared with both treatment modalities over a course of 4 to 6 wk, whereas even after 14 to 22 wk Thy-1+DEC were present only in negligible numbers. Similar to high-dose UV-B or PUVA therapy, administration of GCS resulted in the disappearance of both anti-Thy-1- and anti-Ia-reactive cells. Ultrastructural studies disclosed, however, that these steroid-induced alterations in the surface characteristics were accompanied by a dramatic reduction of the LC population but were not paralleled by morphologic changes of Thy-1+DEC. In the course of 7 wk after cessation of steroid treatment, the number of both Ia+EC and Thy-1+DEC had returned to normal values. The selective removal of either of these two dendritic epidermal cell populations by physicochemical agents may provide an excellent strategy to further clarify the functional properties of both LC and Thy-1+DEC.     

Enrichment and characterization of mouse putative epidermal stem cells

Epidermis, a continuously renewing tissue, is maintained by stem cells that proliferate and replenish worn out or damaged cells in the tissue during life. Cultured epidermal stem cells have great potential in scientific research and clinical application. However, isolating a pure and viable population of epidermal stem cells and culturing them has been challenging. In this study, putative epidermal stem cells of mouse were isolated by combining Hoechst 33342 and propidium iodide staining with fluorescence-activated cell sorting. Molecular markers expression pattern analysis showed that cytokeratin 14, integrin beta1 and p63 are expressed in the sorted putative stem cells, but not active beta-catenin, nestin and involucrin. Our results provide further supporting data that mouse putative epidermal stem cells could be successfully isolated by combining Hoechst dye staining with fluorescence-activated cell sorting and cultured in vitro. The cultured mouse putative epidermal stem cells could be used as a potent tool for studying stem cell biology and testing stem cell therapy.     

Interleukin 2 receptors on cultured murine epidermal Langerhans cells

Rat monoclonal antibodies 3C7 and 7D4 detect two distinct functional regions of the murine interleukin 2 (IL 2) receptor. When studying the emergence kinetics of IL 2 receptors in mixed epidermal cell (EC)-lymphocyte cultures by using 3C7 and 7D4 in an indirect immunofluorescence assay, we regularly encountered a distinctive membrane fluorescence not only on lymphocytes, but also on a subpopulation of cells exhibiting a dendritic morphology. Reasoning that these 3C7/7D4-reactive dendritic cells might represent a subpopulation of epidermal dendritic cells, we studied mouse EC for the presence of 3C7/7D4- reactive cells. Although 3C7/7D4 reactivity was never detected on freshly isolated EC or on epidermal sheets, a small number of 3C7/7D4+ cells was encountered after 24 to 48 hr of culture. These cells exhibited a dendritic shape, expressed Ia antigens, lacked Thy-1 antigens, and displayed the ultrastructural features of Langerhans cells (LC) with the notable exception of Birbeck granules. Although after 24 hr, only 20% of Ia+ EC were 3C7/7D4+, the vast majority of LC displayed 3C7/7D4 binding sites after 4 to 5 days of culture. Preincubation of cultured LC-enriched EC with recombinant human IL 2 prevented subsequent 3C7-but not 7D4-binding to these cells. Western blot analysis of 7D4-reactive material of detergent extracts from LC-enriched EC revealed three bands in the same m.w. range as reported for CTLL cells. These results demonstrate that cultured LC express IL 2 receptors and may bear important implications for a better understanding of growth regulation, differentiation, and immunologic functions of LC.