Ultraviolet B radiation suppresses Langerhans cell migration in the dermis by down-regulation of alpha4 integrin

Background/Purpose: Ultraviolet B (UVB) radiation affects the migration and function of epidermal Langerhans cells (LC) and causes immunosuppression of contact hypersensitivity. It is known that LC leaves the epidermis after exposure to UVB. To know the behavior of LC in the dermis after UVB radiation, we studied the effect of UVB radiation on the expression of integrin families on freshly isolated or cultured murine LC. We also examined whether UVB radiation affects the migration of LC to secondary lymphoid tissue chemokine (SLC/6Ckine).
Methods: Integrin expressions of murine LC cultured in epidermal cell suspension were analyzed using flowcytometry. We used murine LC sorted flowcytometrically for binding assay to extracellular matrix and for migration assay to chemokine. Skin explant assay and immnohistochemical staining for 'cords formation' were performed as previously described.
Results: Twenty and 40 mJ/cm² of UVB radiation down-regulated the expression of α4 integrin on 24 h-cultured LC, but not that of α6, β1, or β4 integrin. The number of cultured LC adhered to fibronectin, a ligand for α4 integrin, was decreased after UVB irradiation, while that to laminin, a ligand for α6 integrin, was not influenced. UVB radiation reduced the number of migrating LC to SLC. Furthermore, skin sheet explant experiments showed that UVB radiation inhibited the 'cords' formation in dermal vessels of the 48 h-cultured skin.
Conclusions: These data suggest that UVB radiation may suppress the migration of LC from the dermis to lymphatic vessels. UVB radiation may downregulate the adherence of LC to dermal fibronectin and migration to SLC, and consequently suppress the migration of LC from the UVB-irradiated dermis to lymphatics.     

Immunostimulatory capabilities of highly enriched Langerhans cells in vitro

Langerhans cells (LC) are epidermal antigen-presenting cells capable of inducing allogenic, antigen-specific, and cytotoxic T cell proliferation. Previous studies have examined the dynamics of LC maintained in vitro in crude epidermal cell (EC) suspensions in which the major cell type is the keratinocyte (KC). To avoid the confounding effects of KC and other immunoregulatory cells on LC dynamics in vitro, highly enriched murine LC (85%) were studied, through 72 h of incubation in vitro, for their ability to present alloantigen (in a primary allogenic proliferation assay) and foreign antigen (in a secondary autologous proliferation assay). The results were compared to similar studies using crude EC suspensions. Freshly prepared LC are very poor stimulators of a primary allogenic proliferation response, with a 12- to 16-fold increase in stimulatory capacity by 72 h using panned-enriched and crude EC suspensions, respectively. Similarly, freshly prepared LC are weak stimulators of a secondary autologous proliferation response, with a 2.5- to 6-fold increase in immunostimulatory capability by 72 h. The overall increased stimulatory effect observed with the crude EC suspensions compared to highly enriched LC is most likely attributed to the effect of KC on T cell proliferation, rather than to a maturation effect of KC on LC during the 72 h of in vitro incubation. Using back-scattered electron imaging, the surface density of MHC-class II molecules (Ia) increased three- to fourfold through culture, which parallels the increase in functional ability. This study demonstrates that LC in either a crude or highly enriched cell suspension mature into potent immunostimulatory cells after incubation in vitro with an increased surface expression of Ia molecules. Keratinocytes are not necessary for LC maturation in vitro, but seem to exert some stimulatory effect by enhancing lymphocyte proliferation in the functional assay system.     

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.     

Quantitation of surface antigens on cultured murine epidermal Langerhans cells: rapid and selective increase in the level of surface MHC products

It was recently discovered that murine epidermal Langerhans cells (LC) changed significantly in function and phenotype when maintained in culture. Notably, accessory cell function for primary immune responses increased while cytologic markers like ATPase, nonspecific esterase, and Birbeck granules were lost. To further analyze LC differentiation, we used flow cytometry and a panel of 22 monoclonal antibodies to quantitate changes in surface antigens at the single-cell level. A striking change was a fivefold increase in the amount of Ia antigens (which are expressed on class II MHC products) during the first day of culture. The increase was evident within 3 h and reached a plateau at 15-24 h. Both I-A and I-E products behaved similarly. The increase in Ia was blocked by 1 microgram/ml cycloheximide. Expression of other surface antigens was then monitored on Ia+ LC by two-color flow cytometry. Low levels of class I (H-2D and H-2K) MHC products were detected on freshly isolated LC, and these antigens also increased severalfold during the first day of culture. Fc receptors (identified with the 2.4G2 mAb) and the F4/80 macrophage antigen decreased, as reported previously. Three antigens that were detected in fresh suspensions were expressed at constant levels in culture. These were the C3bi receptor and the pan leukocyte and interdigitating cell antigens. Several leukocyte antigens that were not found initially on LCs did not appear, including B220 anti-B cell, 33D1 anti-dendritic cell, and CD4, CD5, CD8 T-cell specificities. We conclude that the surface of cultured LCs undergoes selective changes in culture. As a result, the cells are rich in Ia and H-2 and have detectable C3bi receptors, but have little or no LFA-1, Ti, CD4, 5, and 8, 33D1, 2.4G2, F4/80, and B220 antigens.     

Antigen-presenting cell function of epidermal cells activated by hapten application

Exposure to haptens initiates a series of immune and inflammatory reactions that cause migration of epidermal Langerhans cells (LC) to draining lymph nodes, antigen processing, and presentation to T cells. In the present study, the antigen-presenting cell (APC) function of epidermal cells (EC) following hapten application was determined using a cell transfer system. This function of EC in inducing contact sensitivity (CS) in the recipient mice appeared as early as 6 h after hapten painting, and reached its maximum at 24 h. The amount of hapten on EC did not correlate with the function, i.e. the amount retained on the cells was greatest immediately after hapten painting and decreased over time. Several experiments were performed to identify the cell type responsible for the APC function. Through immunomagnetic bead separation, the APC function was detected in Ia^? EC, as well as in unfractionated EC from hapten-painted mice. A purified population of Ia⁺ cells (LC) induced CS with much less efficiency than unseparated cells. Depletion of LC by anti-Ia monoclonal antibody (mAb) and complement-mediated lysis did not impair the APC function, whereas it was reduced by the depletion of Thy-1⁺ cells by anti-Thy-1 mAb and complement-mediated lysis. Moreover, adherent cells that were harvested from a 48-h culture of EC obtained from hapten-painted skin, and were free of contaminating LC and γδ T cells, had a strong capacity to induce CS. These findings indicate that keratinocytes (KC) acquire APC function as well as LC, with hapten application. Phenotypically increased expression of intercellular adhesion molecule-1 (ICAM-1) and Thy-1 on EC was observed following hapten application, whereas expression of Ia and B7/BB1 was unaltered. The APC function of EC from hapten-painted skin was dependent on ICAM-1 and Thy-1 expression, as the mAbs for these molecules reduced the capacity to induce CS. These results suggest that hapten application induces not only LC but also KC to mature functionally and become potent APCs, and that these KC exert the APC function complementarily at local sites following the migration of LC with potent APC function to the draining lymph node.     

Antigen processing and presentation by epidermal Langerhans cells. Induction of immunity or unresponsiveness

The seminal observation made 30 years ago that T cells do not discriminate between native and denatured proteins, whereas B cells generally do, can now be explained by the fact that T cells never see antigens in their native conformation and that intact proteins cannot associate simultaneously with MHC molecules and the TCR. This difference in the ability to recognize antigen based on conformational specificity appears to be a consequence of the fact that the T cell sees antigen not free in solution, but on the surface of an APC in association with MHC molecules. The metabolic events that protein antigens undergo within APC, prior to their presentation in an appropriately processed form to T cells, are called antigen processing. The end-product of antigen processing for CD4+ T cells is a relatively short peptide fragment bound to class II MHC molecules on the surface of an APC that can be recognized by the TCR on the T cells. Because this event is difficult to monitor directly, antigen processing can only be assayed in conjunction with the temporally distal event of T-cell activation, manifested ultimately as proliferative responses or lymphokine secretion. In addition to occupancy of the TCR by the peptide/class II complex, several other antigen-nonspecific receptor-ligand interactions between APC and T cells are required for optimal T-cell activation. M phi, B cells, and LC/DC comprise the principal APC for CD4+ T cells. M phi and B cells have been studied extensively with respect to their antigen processing and presenting capacities. Only recently, however, have such capacities been investigated in LC and DC; these studies now indicate freshly isolated LC (but not cultured LC and DC) to possess efficient antigen processing capabilities. In this respect, LC have been proposed to represent evolving (or "maturing") forms of DC: Freshly isolated LC (which retain morphologic and functional properties of epidermal LC in situ) are the equivalent of tissue forms of DC, whereas cultured LC resemble lymphoid or circulating DC. Cultured LC and DC appear to be the sole effective APC for inducing primary T-cell responses in vitro. Possibly underlying this property is the ability of cultured LC and DC (but not M phi, B cells, or freshly isolated LC) to induce formation of T-cell clusters during the course of such responses. The capacity of accessory cells to function as APC varies depending upon the type of APC and T cells examined.(ABSTRACT TRUNCATED AT 400 WORDS)     

Epidermal Langerhans cells contain intermediate-sized filaments of the vimentin type: an immunocytologic study

In epidermal cell suspensions, prepared from healthy human and murine skin, Langerhans cells (LC) were identified by indirect immunofluorescence microscopy using monoclonal antibodies directed against human T6 and Ia markers, and against a murine Iak determinant, respectively. The cells were double-labeled on cytospin slides with antibodies against a cytoskeletal protein, either vimentin or keratin. In this way it was shown that epidermal LC contain intermediate-sized filaments of the vimentin type.     

Direct effects of glucocorticosteroids on epidermal Langerhans cells

To determine the direct effects of glucocorticosteroids on epidermal Langerhans cells (LC), we treated isolated LC with dexamethasone (DEX) in vitro, and investigated Ia expression by LC using immunofluorescence microscopy and FACS analysis. We found that DEX directly decreased the number of Ia+ LC in a dose- and time-dependent manner. Pulse incubation with DEX also inhibited the immunostimulatory function of LC in vitro. FACS analysis demonstrated that LC detected in DEX-treated culture expressed a similar amount of Ia antigen and Fc receptor on the cell surface as LC cultured with the solvent control, suggesting that LC may be composed of a heterogeneous population in terms of sensitivity to DEX, and DEX may completely abolish the expression of surface molecules on a subpopulation of LC or may be cytolytic to this sensitive population.     

Effective enrichment of murine epidermal Langerhans cells by a modified(mismatched) panning technique.

A method for the enrichment of murine epidermal Langerhans cells (LC) is described in detail. It is based on positive selection of LC from pre-enriched fresh or cultured epidermal cell suspensions derived from ear skin by a modified panning technique. The method uses the interspecies cross-reactivities of anti-immunoglobulin antibodies: when LC in an epidermal cell suspension are labeled with mouse anti-major histocompatibility complex (MHC) class II antibodies they bind to petri dishes coated with anti-rat immunoglobulin antibodies. We therefore call this method "mismatched panning." After rinsing off non-adherent cells, the adherent LC can easily be dislodged by adding excess amounts of rat immunoglobulins, which effectively compete with the LC-bound mouse anti-MHC class II antibodies for binding to the petri dish. Using this modified panning technique, both fresh and cultured LC could be enriched up to more than 90% purity. From one ear, 2.0-3.0 x 10(4) fresh LC and 3.0-4.5 x 10(4) cultured LC could be obtained. Of all LC present in a primary, unenriched epidermal cell suspension, 40-60% were recovered when panned immediately after isolation of the epidermal cells and 50-75% when panned after 3 d of epidermal cell culture. Viability of panned LC was consistently more than 90%. Antigen presenting and T-cell-stimulating capacity of LC and responses to the cytokines granulocyte/macrophage colony-stimulating factor and tumor necrosis factor-alpha were not impaired by this panning procedure. The major advantage of this method compared to pre-existing panning techniques is the ease with which adherent LC can be dislodged from the panning dishes. Because the elution procedure is very gentle, virtually all panned LC are viable. As a consequence, good yields of highly enriched LC can be obtained in a reasonable time.     

Spleen dendritic cells exhibit altered morphology and increased allostimulatory capacity after short-term culture.

Ia-bearing dendritic cells (DC) are a class of bone marrow-derived antigen-presenting cells that appear to possess an increased capacity to stimulate resting T lymphocytes. DC from different tissues share several morphologic, phenotypic and functional attributes. For example, freshly isolated DC from spleen resemble phenotypically and functionally freshly isolated Langerhans cells (LC) from epidermis; in addition, during short-term culture both DC and LC undergo several parallel changes including modifications affecting phenotype, capacity to present protein antigens, and ability to route surface Ia molecules into intracellular acidic compartments (J Immunol 1990: 145: 2820-2826). In the present study we show, using immunoelectron microscopy with anti-Ia and anti-33D1 monoclonal antibodies, freshly isolated DC in suspension to have a smooth cell surface with few and short cytoplasmic projections. By contrast, cultured DC display conspicuous bulbous cytoplasmic protrusions. In addition, spleen DC following culture for 24-48 hours exhibit an increased ability to stimulated allogeneic T lymphocytes in the primary mixed leukocyte reaction. These changes, similar to those described for freshly isolated and cultured LC respectively, further substantiate the close relationship between DC and LC.     

Immunologic aspects of acute cutaneous graft-versus-host disease: decreased density and antigen-presenting function of Ia+ Langerhans cells and absent antigen-presenting capacity of Ia+ keratinocytes

Cutaneous graft-versus-host disease (GVHD) provides a unique model for studying the pathogenesis of several important lymphocyte-mediated skin diseases. Morphologic studies have suggested that Ia antigen (Ia)-bearing epidermal Langerhans cells (LC) may be specific targets for destruction in these conditions. Keratinocytes synthesize and express Ia in GVHD and some other lymphocyte-mediated skin disorders; Ia+ keratinocytes, constitutively able to secrete epidermal cell-derived thymocyte activating factor (ETAF)/interleukin 1, may possess antigen-presenting capacity, thus leading to enhanced cutaneous immune responses and disease chronicity. We therefore investigated the fate of Ia+ LC, and the potential antigen-presenting capacity of Ia+ keratinocytes, in a murine model of GVHD. Lethally irradiated C3H/He (H-2k) mice developed acute cutaneous GVHD, and expressed keratinocyte Iak, 8 days after injection of BALB/c (H-2d) bone marrow and spleen cells. Immunofluorescence studies showed a progressive decrease in the density of Ia+ epidermal LC during the evolution of GVHD. This decrease was paralleled by a progressive reduction in the allostimulatory capacity of GVHD epidermal cells (EC) in the allogeneic EC-lymphocyte reaction (ELR). The fall in the density of Ia+ LC, and in EC allostimulatory capacity in both primary and secondary ELRs, was consistently greater in GVHD mice than in mice treated only with x-irradiation. The allostimulatory capacity of GVHD and x-irradiated EC could not be restored by addition of indomethacin or exogenous ETAF to ELR cultures. The decreased allostimulatory capacity was not the result of inhibition of the ELR, since EC from GVHD and x-irradiated mice did not cause suppression when added to control ELR cultures. The capacity of EC to present ovalbumin, purified protein derivative of tuberculin, 2,4,6-trinitrobenzenesulfonic acid coupled to EC, and native cytochrome c (CYTc) to antigen-specific T-cell lines, clones, or hybridomas was reduced in x-irradiated mice and markedly decreased in GVHD mice. The capacity of EC from x-irradiated and GVHD mice to present CYTc fragment 81-104, which does not require further processing or catabolism by accessory cells, was similarly decreased. Taken together, the results indicate that: the function of LC is markedly and progressively impaired in acute GVHD; LC function is also decreased, but to a lesser extent, following x-irradiation alone; and Ia+ keratinocytes from lethally irradiated mice undergoing GVHD do not exhibit antigen-presenting capacity.     

Internalization and acidification of surface HLA-DR molecules by epidermal Langerhans cells: a paradigm for antigen processing

CD4+ T lymphocytes recognize multi-molecular complexes, formed by major histocompatibility complex class II molecules and exogenous antigens, on the surface of antigen-presenting cells (APC). For most protein antigens, processing is required to produce immunogenic peptide fragments that can then form stable associations with class II molecules. These two processes, the modification of antigen and its coupling to class II molecules, are thought to occur in acidic endosomal compartments. Furthermore, membrane class II molecules are endocytosed in APC and may provide ligands for the immunogenic peptides. To gain insight into these processes, we examined the internalization and acidification of membrane HLA-DR molecules by three APC populations: 1) freshly isolated Langerhans cells (LC), 2) LC after 48-72 h of bulk epidermal cell culture, and 3) peripheral blood monocytes (PBM). Using FITC-conjugated anti-HLA-DR monoclonal antibodies (MoAb), endocytosis was studied by fluorescence microscopy and by flow cytometry (pulse width analysis), while acidification was assessed by exploiting the pH sensitivity of fluorescein fluorescence. We observed both freshly isolated LC and PBM to internalize surface HLA-DR molecules into acidic compartments with great efficiency. Endocytosis was inhibited by the addition of azide and 2-deoxy-D-glucose, whereas acidification was partially blocked by treatment with ammonium chloride or chloroquine. The degree of internalization and acidification of HLA-DR molecules was greatly influenced by the degree of Ab cross-linking. On the other hand, cultured LC were capable of internalizing HLA-DR molecules, but were not able to acidify the environments to which these molecules were delivered; this loss of acidification capacity was partially restored by treatment with phorbol 12-myristate 13-acetate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression, but lack of calcium mobilization by high-affinity IgE Fcε receptor I on human epidermal and dermal Langerhans cells

Abstract In atopic dermatitis (AD) patients, IgE molecules are demonstrated on the surface of Langerhans cells (LC). FcεRI molecules, which are present on the surface of LC in AD patients as well as normal individuals, are responsible for this binding. In this study, we have investigated phenotypic and functional characteristics of FcεRI on epidermal and dermal cell populations. Epidermal and dermal cell suspensions were prepared enzymatically with dispase followed by either trypsin or collagenase treatment, respectively. Peripheral blood basophils were negatively selected by excluding other leukocytes with surface marker staining. Consistent with previous reports, both peripheral blood basophils and epidermal LC were positively stained with anti FcεRI monoclonal antibody. In addition, an FcεRI positive population was demon-strated among dermal HLA-DR positive cells. These cells express significant amounts of HLA-DR molecules (DR^Hi) and co-express CD la molecules, which identifies them as LC-like dendritie APC of the dermis. No other FcεRI positive population was found in the other dermal DR^Mid or DR populations, except for a minor DR^lo population, presumably mast cells. To analyze whether these FcεRI molecules are signal transducing for LC, intracellular calcium mobilization after crosslinking of FcεRI was measured with How cytometry. Following crosslinking, peripheral blood basophils clearly increased intracellular calcium. On the other hand, neither normal epidermal LC nor dermal DR^HiCD Ia⁺ cells changed their intracellular calcium level after FcεRI crosslinking. These data indicate that normal epidermal and dermal LC, but not basophils, are resistant to calcium flux following FcεRI engagement.     

Review of hair follicle dermal cells

Hair follicle stem cells in the epithelial bulge are responsible for the continual regeneration of the hair follicle during cycling. The bulge cells reside in a niche composed of dermal cells. The dermal compartment of the hair follicle consists of the dermal papilla and dermal sheath. Interactions between hair follicle epithelial and dermal cells are necessary for hair follicle morphogenesis during development and in hair reconstitution assays. Dermal papilla and dermal sheath cells express specific markers and possess distinctive morphology and behavior in culture. These cells can induce hair follicle differentiation in epithelial cells and are required in hair reconstitution assays either in the form of intact tissue, dissociated freshly prepared cells or cultured cells. This review will focus on hair follicle dermal cells since most therapeutic efforts to date have concentrated on this aspect of the hair follicle, with the idea that enriching hair-inductive dermal cell populations and expanding their number by culture while maintaining their properties, will establish an efficient hair reconstitution assay that could eventually have therapeutic implications.     

The role of dendritic cells in cutaneous immunity

This article reviews the role of dendritic cells in cutaneous immunity. Langerhans cells (LC) found in the epidermis are the best-characterized dendritic cell population. They have the ability to process antigen in the periphery, transport it to the draining lymph nodes (DLN) where they are able to cluster with, and activate, antigen-specific naive T cells. During migration LC undergo phenotypic and functional changes which enable them to perform this function. There are other less well-characterized dendritic cells including dendritic epidermal T cells, dermal dendrocytes and dermal ‘LC-like’ cells. Although there is no evidence that dendritic epidermal T cells (DETC) can present antigen or migrate to lymph nodes, they do influence the intensity of cutaneous immune responses to chemical haptens. Antigen-presenting cells (APC) in the dermis may provide alternative routes of antigen presentation which could be important in the regulation of skin immune responses. Therefore, dendritic cells are vital for the induction of immune responses to antigens encountered via the skin. LC are particularly important in primary immune responses due to their ability to activate naive T cells. The faster kinetics of secondary responses, and the ability of nonprofessional APC to induce effector function in previously activated cells, suggest that antigen presentation in the DLN may be less important in responses to previously encountered antigens. In these seondary responses, dendritic and nondendritic APC in the skin may directly induce effector functions from antigen-specific recirculating cells. Received: 24 April 1995     

Effects of ultraviolet radiation on murine epidermal Langerhans cells: doses of ultraviolet radiation that modulate ICAM-1 (CD54) expression and inhibit Langerhans cell function cause delayed cytotoxicity in vitro.

Low doses (100 J/m2) of ultraviolet B (UVB) radiation from sunlamp fluorescent FS20 tubes inhibit the ability of freshly isolated murine epidermal Langerhans cells (LC) to support anti-CD3 MoAb-induced T-cell mitogenesis and selectively inhibit the upregulation of ICAM-1 expression by LC without causing appreciable cytotoxicity in short-term (less than or equal to 24 h) incubations (J Immunol 146:3347-3355, 1991). In the present study, epidermal cells (EC) were exposed to UVB radiation or were sham-irradiated and cultured for 24, 48, or 72 h when LC were recovered, enumerated, and assayed for simultaneous expression of I-A antigens and ICAM-1 by flow cytometry. UVB-irradiated LC that had been cultured for 24 h exhibited levels of I-A antigens comparable to those on unirradiated LC but expressed substantially less ICAM-1. After 48 and 72 h, cultured UVB-irradiated LC expressed somewhat lower levels of I-A antigens and markedly less ICAM-1 than unirradiated controls. Although similar numbers of LC were recovered from cultures initiated with UVB-irradiated and unirradiated epidermal cells after 24 h, far fewer identifiable LC were recovered from cultures seeded with irradiated cells at 48 and 72 h (approximately 50 and approximately 10% of control, respectively). The effect of UVB radiation on the survival of LC in vitro was not reversible with exogenous TNF alpha (125 U/ml) alone or granulocyte/macrophage colony-stimulating factor (5 ng/ml) and IL-1 (50 U/ml) in combination, although these cytokines had modest effects on the expression of I-A antigens and ICAM-1 by cultured UVB-irradiated LC. Results of survival studies performed with enriched LC preparations demonstrated that UVB radiation was clearly cytotoxic for LC and did not merely downregulate surface expression of I-A antigens or alter LC buoyant density. Exposure of LC to radiation from blacklight fluorescent (UVA) tubes (0.25 J/cm2) in the presence of 8-methoxypsoralen (1 micrograms/ml; PUVA) or monochromatic UVC radiation (20 J/m2) also inhibited LC accessory cell function. Results of survival studies performed with EC that had been exposed to PUVA or UVC radiation before culture were similar to those of studies performed with UVB-irradiated cells, although PUVA- and UVC-induced LC cytotoxicity was much more pronounced 48 h after culture initiation than UVB-induced cytotoxicity. UVA radiation alone augmented LC recovery at 24 and 48 h, but did not influence I-A antigen or ICAM-1 expression.(ABSTRACT TRUNCATED AT 400 WORDS)     

Decreased expression of epidermal cytoplasmic antigens in cultured human keratinocytes

The expression of upper cytoplasmic (U-CYT) antigens which are expressed only in the superficial layers of the epidermis and are markers of epidermal cell differentiation in vivo and of basement zone (BMZ) antigens reacting with bullous pemphigoid serum was studied in keratinocytes in tissue culture. The cells were cultured at an acid pH (5.6-5.8) similar to that of skin and without feeder cells, dermal tissue, or collagen. It was found that the expression of U-CYT antigens decreased markedly in culture. These antigens were expressed in 45-65% of epidermal cells prepared from fresh skin, but in only 5-10% of cells which had been grown in primary culture over 1 mo, and in no cells in secondary or tertiary culture. By contrast, BMZ antigens continued to be expressed in culture. These antigens were expressed by 20-35% of epidermal cells prepared from fresh tissue and by 15-35% of keratinocytes in primary, secondary or tertiary culture. These findings indicate that U-CYT and BMZ antigens can be used to type subpopulations of human keratinocytes in suspension, and suggest that the differentiation of these cells in vitro differs from that which occurs in vivo.     

Interleukin 1β and the stimulation of Langerhans cell migration: comparisons with tumour necrosis factor α

Epidermal Langerhans cells (LC) and the cells into which they mature are believed to play a pivotal role in cutaneous immune function. The induction phase of contact sensitization is associated with the migration of LC from the skin and their accumulation as dendritic cells (DC) in lymph nodes draining the site of exposure. We have demonstrated previously that tumour necrosis factor α (TNF-α), an epidermal cytokine produced by keratinocytes, provides one signal for LC migration. We describe here experiments designed to evaluate the influence of interleukin 1β (IL-1β), a product exclusively of LC in murine epidermis, on LC migration, LC morphology and DC accumulation, and to compare the effects of this cytokine with those of TNF-α. Both cytokines induced a significant reduction in the frequency of epidermal LC and the arrival of DC in draining lymph nodes. Changes in both parameters were induced more rapidly following intradermal administration of TNF-α than were observed after treatment with IL-1β. However, the reduction in LC frequency was more persistent with IL-1β. Both cytokines caused the activation of LC, characterized by the acquisition of a more dendritic morphology and the increased expression of Ia molecules. These results demonstrate that IL-1β and TNF-α can each stimulate the migration of epidermal LC, but that the changes induced by these cytokines are not identical. Received: 9 September 1996     

Cultured human Langerhans cells resemble lymphoid dendritic cells in phenotype and function

Freshly isolated murine epidermal Langerhans cells (LC) are weak stimulators of resting T cells. Upon culture their phenotype changes, their stimulatory activity increases significantly, and they come to resemble lymphoid dendritic cells. Resident murine LC, therefore, might represent a reservoir of immature dendritic cells. We have now used enzyme cytochemistry, a panel of some 80 monoclonal antibodies, and immunofluorescence microscopy or two-color flow cytometry, as well as transmission electron microscopy, to analyse the phenotype and morphology of human LC before and after 2-4 d of bulk epidermal cell culture. In addition, LC were enriched from bulk epidermal cell cultures, and their stimulatory capacity was tested in the allogeneic mixed leukocyte reaction and the oxidative mitogenesis assay. Cultured human LC resembled human lymphoid dendritic cells in morphology, phenotype, and function. Specifically, LC became non-adherent upon culture and developed sheet-like processes (so-called "veils"), decreased their surface ATP/ADP'ase activity, and lost nonspecific esterase activity. As in the mouse, surface expression of MHC class I and II antigens increased significantly, and FcII receptors were significantly reduced. Markers that are expressed by dendritic cells (like CD40) appeared on LC following culture. Cultured human LC were potent T-cell stimulators. Our findings support the view that resident human LC, like murine LC, represent immature precursors of lymphoid dendritic cells in skin-draining lymph nodes.     

Characterization of cutaneous antigen presentation in partially inbred miniature swine

Abstract MHC class I and II-defined, partially inbred miniature swine have recently become available as a large animal model in transplantation immunology. To investigate cutaneous immunocompetence in this model, cutaneous antigen presenting cell (ARC) function was assessed. For morphologic analysis, punch biopsies were examined by electron microscopy. By this technique, epidermal Langerhans cells bearing typical Birbeck granules could be detected. For functional studies, epidermal cell (EC) suspensions were prepared from split thickness skin specimens. Using FACS analysis, freshly prepared epidermal cell suspensions contained 1.8-4.7% MHC class II-positive cells. These EC potently stimulated allogeneic nylon wool-enriched peripheral blood T cells in the primary mixed EC-lymphocyte reaction. For in vivo assessment of cutaneous APC function. EC suspensions enriched for or depleted of class II-positive EC were generated by panning of class II-positive EC using mouse anti-MHC class II antibodies and anti-mouse IgG-coatcd petri dishes. EC were then coupled to the hapten trinitrophenol (TNP) and injected s.c. into autologous or MHC-mismatched pigs twice at a one week interval. One week later, pigs were challenged by s.c.-injection of 0.5-1 × 10⁷ TNP-coupled or uncoupled EC. Autologous unseparated EC as well as EC enriched for MHC class II-positive cells were able to sensitize naive animals against TNP, whereas neither TNP-coupled EC depleted of class II-positive APC, MHC-mismatched EC coupled to TNP, nor uncoupled EC induced immunity to TNP. Our data indicate that inbred miniature swine possess competent cutaneous APC which are able to induce cutaneous immunity in a manner similar to Langerhans cells in murine or human skin.