Doses of ultraviolet radiation that modulate accessory cell activity and ICAM-1 expression are ultimately cytotoxic for murine epidermal Langerhans cells.

Mechanisms that underlie immunomodulatory properties of ultraviolet (UV) radiation remain incompletely characterized. Recently, we have studied effects of UV on the functional activity of epidermal Langerhans cells (LC) and have attempted to relate inhibitory effects of UV on LC function to modulatory effects of UV on adhesion molecule expression by LC. Exposure of LC in vitro to amounts of UVB, UVC, or psoralen+UVA (PUVA) radiation that inhibited LC function also prevented increased expression of intercellular adhesion molecule-1 by LC in vitro. Subsequent studies revealed that amounts of UV radiation that inhibited LC function and modulated ICAM-1 expression also decreased LC survival in vitro, although UV-induced LC cytotoxicity did not become apparent until 48-72 h after UV exposure. Our results are consistent with those of previous studies that suggested that low doses of UV radiation were cytotoxic for LC in situ. The potential cytotoxicity of UV radiation for LC should be considered when studies of effects of UV radiation on immune responses in skin are interpreted.     

Contact allergens modulate the expression of MHC class II molecules on murine epidermal Langerhans cells by endocytotic mechanisms.

MHC class II molecules play an important role during the sensitization phase of allergic contact dermatitis. To study the influence of contact allergens on the expression of these molecules by murine epidermal Langerhans cells (LC), we performed a flow-cytofluorometric analysis of the Ia-antigen expression after in vivo application of contact allergens. A distinct decrease in the Ia-antigen expression of the entire LC population was noticed 3 h after in vivo application of the contact allergen 2,4-dinitrofluorobenzene (DNFB). This decrease was transient and balanced 24 h after in vivo application of DNFB. A downregulation was also detectable after in vivo application of the contact allergens 1-chloro-2,4-dinitrobenzene (DNCB), oxazolone, K2Cr2O7, 2,4,6-trinitrochlorobenzene (TNCB), and toxic concentrations of the irritant compound sodium dodecyl sulfate (SDS). In vitro studies showed that freshly prepared as well as 3-d cultured LC downregulated their Ia-antigen expression in the presence of DNFB, which was used as a model compound. This decrease was not inhibited by the MHC class II molecule transport-inhibitor brefeldin A nor by the ionophore monensin. The inhibition of receptor-mediated endocytosis with hypertonic media (0.45 M sucrose) abolished the DNFB-mediated downregulation of Ia-antigen expression. An accelerated clearance of cell-surface-expressed antibody-labeled IA molecules was detectable in the presence of DNFB. Internalization studies carried out with peroxidase-labeled anti-IA-antibody complexes showed remarkable alterations in the intracellular distribution of endocytosed material under the influence of subtoxic concentrations of DNFB, DNCB, K2Cr2O7, and TNCB. The irritant substance sodium dodecyl sulfate (SDS) influenced the intracellular distribution pattern of internalized material only when used in toxic concentrations. An augmented participation of MHC class II molecules in endocytotic processes is mediated by reactive substances like contact allergens and might contribute to the processing and presentation of these compounds.     

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.     

Effects on murine epidermal Langerhans cells of drugs known to cause recrudescent herpes simplex virus infection in a mouse model.

A number of agents have been shown to alter the latent state of herpes simplex virus in murine sensory ganglia. However, it seems that effective triggers of recrudescent disease must act not only to reactivate latent HSV infection, but also to create a favorable environment in the skin for viral replication. The possibility that alteration of the local Langerhans cell population is one way in which effective triggers of recrudescence may act has been investigated. Of the agents tested, which affect latent HSV, only DMSO significantly altered the numbers of ATPase-bearing Langerhans cells in the epidermis, maximally reducing their density by 83% in 48 h. Xylene and retinoic acid had no discernible effect on numbers of ATPase-staining cells over the 4 d tested. However, the extent to which agents reduced ATPase-staining cell numbers did not correlate with their ability to affect the antigen-presenting capacity of the cells in HSV-specific T-cell proliferative assays in vitro. Xylene and retinoic acid markedly reduced the accessory cell function of epidermal cell suspensions, whereas DMSO had no effect.     

Adhesion molecules CD11a, CD18, and ICAM-1 on human epidermal Langerhans cells serve a functional role in the activation of alloreactive T cells.

Binding of antigen-presenting cells (APC) to T cells via adhesion molecules is thought to deliver accessory signals that are required for efficient T-cell activation. To determine whether Langerhans cells (LC) express relevant adhesion molecules on their surfaces, we employed two-color immunofluorescence. Human epidermal cells (EC), Ficoll-enriched for LC (greater than 10%), were incubated with monoclonal antibodies (MoAb) specific for the adhesion molecules CD11a (LFA-1 alpha), CD18 (LFA-1 beta), or ICAM-1; staining was evaluated by fluorescence microscopy. After 12 h of culture only HLA-DR+ cells (LC) expressed CD11a, CD18, and ICAM-1. As a test for the functional relevance of such adhesion molecule expression, we examined the capacity of the above MoAb to block LC stimulation of alloreactive T cells: EC were co-cultured with allogeneic peripheral blood mononuclear leukocytes (PBML) for 5 d in the presence or absence of MoAb; proliferation was measured by [3H]-thymidine uptake. MoAb against CD11a, CD18, or ICAM-1 reduced the allostimulatory capacity of LC by greater than 70%; combinations of these MoAb reduced proliferation even more (90%). We conclude that interaction of adhesion molecules on LC with ligands on T cells is required for optimal allo-antigen-dependent T-cell activation, perhaps by delivering accessory signals.     

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.     

Examination of tetrachlorosalicylanilide (TCSA) photoallergy using in vitro photohapten-modified Langerhans cell-enriched epidermal cells.

Lymphocytes from BALB/c mice photosensitized in vivo to tetrachlorosalicylanilide (TCSA) were investigated to determine whether they could be stimulated to proliferate when cultured with Langerhans cell-enriched cultured epidermal cells (LC-EC) photohapten-modified in vitro with TCSA + UVA radiation. Cultured LC-EC were photohapten-modified in vitro by irradiation in TCSA-containing medium using a 1000-watt solar simulator equipped with filters to deliver primarily UVA radiation (320-400 nm). Lymphocytes from TCSA-photosensitized mice were incubated with LC-EC that had been treated in vitro with 0.1 mM TCSA and 2 J/cm2 UVA radiation (TCSA + UVA). Responder lymphocytes demonstrated a significant increase in their blastogenesis response compared to lymphocytes that were incubated with LC-EC irradiated with UVA prior to treatment with TCSA (UVA/TCSA) or with LC-EC that had received no treatment. Lymphocytes from naive mice or mice photosensitized with musk ambrette (MA) demonstrated a significantly lower response to LC-EC modified with TCSA + UVA, indicating the specificity of the response. Maximum blastogenesis response was achieved when LC-EC were treated with 0.1 mM TCSA and a UVA radiation dose of at least 0.5 J/cm2. Epidermal cells depleted of LC by treatment with anti-Ia antibody plus complement or by an adherence procedure were unable to stimulate this blastogenesis response. Epidermal cells treated in vitro with TCSA + UVA demonstrated enhanced fluorescence compared to control cells. The fluorescence observed was not restricted to any specific epidermal cell type; however, fluorescence microscopy studies revealed that dendritic Ia-positive cells, presumably LC, were also TCSA fluorescent. Flow cytometry showed that Ia-positive epidermal cells demonstrated the greatest UV fluorescence when treated with TCSA + UVA compared to both cells irradiated with UVA and subsequently treated with TCSA and untreated cells. This is consistent with the enhanced antigen presentation capability of TCSA + UVA treated LC-EC, which leads to the conclusion that LC photohapten-modified in vitro with TCSA + UVA demonstrate enhanced TCSA fluorescence and are capable of stimulating lymphocytes from TCSA photosensitized mice in an antigen-specific manner.     

Gamma-interferon promotes the release of IgE-binding factors (soluble CD23) by human epidermal Langerhans cells.

In a previous study, we have demonstrated that human epidermal Langerhans cells (LC) are induced to express Fc epsilon R2/CD23 by stimulation with IL-4 and/or IFN-gamma. In this study, using LC-enriched and LC-depleted epidermal cell (EC) cultures, we have shown that stimulation with IL-4 and/or IFN-gamma not only led to Fc epsilon R2/CD23 expression on normal human LC but also to the release of significant amounts of IgE-BF (soluble CD23). Furthermore, stimulation with IL-4 was more effective in induction of Fc epsilon R2/CD23 on LC when compared to IFN-gamma, which, in contrast, strongly promoted the release of IgE-BF. These results indicate that Fc epsilon R2/CD23-positive LC represent a potential source of IgE-BF and that, as is observed in monocytes or U937 cells, IFN-gamma and IL-4 differently regulate the Fc epsilon R2/CD23 expression and release on LC.     

The ICAM-3/LFA-1 interaction is critical for epidermal Langerhans cell alloantigen presentation to CD4+ T cells

Intercellular adhesion molecule (ICAM)-3 is a recently described member of the immunoglobulin superfamily and, as such, is closely related to ICAM-1 and ICAM-2. All three ICAMS are cognate for the counter-receptor lymphocyte function associated antigen-1 (LFA-L CD11a/CD18). Unlike ICAM-1 and ICAM-2. ICAM-3 is constitutively expressed at high levels on resting leucocytes. We investigated the expression and function of ICAM-3 in normal skin (n= 5), as well as its expression in psoriasis (n= 4). atopic eczema (n= 4), allergic (rhus) contact dermatitis (n=3). and cutaneous T-cell lymphoma (CTCL. n=2). Five-micrometre cryostat sections of skin were stained using monoclonal antibodies to ICAM-3 and A well characterized immunoperoxidase technique. In normal skin. ICAM-3 was expressed by all cutaneous leucocytes hut most striking was the strong expression of ICAM-3 by Langerhans cells within both epidermis and dermis. This observation was confirmed by double-labelling with CD1a and negative staining with an IgG1 isotype control. In psoriasis, atopic eczema, allergic contact dermatitis, and CTCL. ICAM-3 was co-expressed on all CD1a⁺ cells, although, in psoriasis, the intensity of ICAM-3 expression was reduced. Functional blocking experiments were performed to determine whether the observed ICAM-3 expression on Langerhans cells was functionally important in antigen presentation. CD4⁺ T cells were prepared from peripheral blood and 10⁵ CD4⁺ T cells combined with 10⁵ epidermal cells harvested from keratome biopsies of normal skin of an individual allogeneic to the T-cell donor. Addition of 50 μg anti-ICAM-3 to the co-culture resulted in a consistent (50%) reduction in degree of alloantigen presentation by Langerhans cells to T cells. Inhibition was 77% of that produced by the addition of anti-LFA-1. These data indicate that ICAM-3 is constitutively expressed by Langerhans cells and is a major ligand for LFA-1 on CD4⁺ T cells during their response to Langerhans cells. Because fresh Langerhans ceils constitutively express little ICAM-1. whereas ICAM-3 is constitutively expressed at high levels, it would appear that 1CAM-3 is the dominant functional ICAM on in situ Langerhans cells in the normal epidermis.     

In situ expression of the costimulatory molecules CD80 and CD86 on Langerhans cells and inflammatory dendritic epidermal cells (IDEC) in atopic dermatitis

Abstract The functional expression of costimulatory molecules on antigen-presenting cells may be a key event in the pathogenesis of atopic dermatitis (AD). Recently, the expression of CD86 (B7-2/B70) has been demonstrated on CD1a⁺ epidermal dendritic cells (DC) in AD lesions by immunohistological and functional analysis. Therefore, we sought to further characterize the in situ expression of costimulatory molecules on these cells, considering the two subpopulations of (1) CD1a⁺⁺⁺/CD11b^– Langerhans cells (LC) containing Birbeck granules and (2) CD1a⁺/CD11b⁺⁺⁺ inflammatory dendritic epidermal cells (IDEC), devoid of Birbeck granules, from AD and other inflammatory skin diseases. Flow cytometry, skin mixed lymphocyte reactions (SMLR) and immunohistological analysis were performed, and showed that IDEC and not LC are the relevant cells expressing the costimulatory molecules CD80 and CD86 in situ. This expression varied with the underlying diagnosis, with AD showing the highest expression of both CD80 and CD86 in situ. Furthermore, the expression of CD80, CD86 and CD36 were significantly correlated. With short-term culture, both CD80 and CD86 were further upregulated on LC and IDEC. Finally, anti-CD86 antibody reduced the stimulatory activity of epidermal DC. These results indicate that costimulatory molecules on LC and IDEC might play a role in the pathogenesis of AD. Received: 7 June 2000 / Accepted: 21 January 2001     

Granulocyte macrophage--colony-stimulating factor (GM-CSF) decreases CD1a expression by human Langerhans cells and increases proliferation in the mixed epidermal cell-lymphocyte reaction (MELR).

Langerhans cells (LC) undergo a variety of phenotypic and functional changes in vitro. To determine the effects of granulocyte macrophage--colony-stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-alpha), and interleukin 1-alpha (IL-1) on LC phenotype in vitro, epidermal cell suspensions were enriched for LC by density-gradient centrifugation and cultured in the presence of 10 ng/ml of these cytokines. The percentage of cells expressing the surface protein CD1a was determined by flow cytometry. This percentage typically dropped after 48 h culture in both control and cytokine-treated medium to less than half that of the starting value. By the fifth day, the percentage of cells expressing CD1a in TNF-alpha and IL-1--treated cultures was still near half of the starting value, slightly above that of control cultures. Treatment with GM-CSF caused large and consistent decreases in the percentage of epidermal cells expressing CD1a. Cell viability in each of the three cytokine-treated cultures was identical to the control cultures, with essentially all cells having died by the sixth day after isolation. To determine the functional effects of these cytokines, the cytokine-containing medium was replaced after 72 h with medium containing purified allogeneic T cells and proliferation measured. Preliminary experiments showed no increased proliferation induced by IL-1 or TNF-alpha--treated epidermal cells. GM-CSF-treated epidermal cells induced 2-3 times more T-cell proliferation than epidermal cells cultured without additional cytokines. We conclude that GM-CSF, a cytokine known to be produced by keratinocytes in vitro, decreases CD1a expression by human LC and increases their ability to stimulate proliferation by allogeneic T cells.     

Keratinocyte apoptosis induced by ultraviolet B radiation and CD95 ligation -- differential protection through epidermal growth factor receptor activation and Bcl-x(L) expression.

Previous work has shown that activation of the epidermal growth factor receptor by endogenous or exogenous signals markedly enhances survival of cultured keratinocytes upon cellular stress such as passaging. This is due, in part, to epidermal-growth-factor-receptor-dependent expression of Bcl-x(L), an antiapoptotic Bcl-2 homolog. In this study we tested whether epidermal-growth-factor-receptor-dependent signal transduction and attendant Bcl-x(L) expression affected survival of human keratinocytes upon exposure to a frequently encountered apoptotic stimulus, radiation with ultraviolet B. We describe that blocking epidermal-growth-factor-receptor-dependent signal transduction sensitized normal keratinocytes to undergo apoptosis upon ultraviolet B radiation with solar light characteristics. Forced expression of Bcl-x(L) partially but significantly inhibited ultraviolet-B-induced apoptosis of immortalized keratinocytes (HaCaT). Bcl-x(L) overexpression afforded no protection to HaCaT cells against apoptosis induced by binding of an agonist antibody to the death receptor CD95, however. CD95 activation has previously been shown to functionally contribute to apoptosis in ultraviolet-irradiated keratinocytes. These results indicate that epidermal growth factor receptor activation and attendant Bcl-x(L) expression provided a physiologically relevant protective pathway of keratinocytes against ultraviolet-induced but not CD95-dependent apoptosis.     

The expression of endothelial leukocyte adhesion molecule-1 (ELAM-1), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) in experimental cutaneous inflammation: a comparison of ultraviolet B erythema and delayed hypersensitivity.

Endothelial cell adhesion molecule-1 (ELAM-1), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) are cytokine-regulated cell surface molecules involved in leukocyte adhesion. We have studied two forms of cutaneous inflammation to investigate in vivo the kinetics of adhesion molecule expression in relation to tissue accumulation of leukocytes. Immunohistology was performed on skin biopsies taken from human volunteers at 1, 6, 24, 72 h, and 1 week after two minimal erythema doses (MED) of ultraviolet B (UV-B) or intra-cutaneous tuberculin-purified protein derivative (PPD) (10-100 U). ELAM-1 expression on vascular endothelium and polymorphonuclear leukocyte infiltration were first observed at 6 h and maximal at 24 h after both UV-B and PPD. At 72 h and 1 week, however, endothelial ELAM-1 was more strongly expressed in PPD biopsies. VCAM-1 was minimally expressed in control skin, and was induced above background levels on endothelium, on some perivascular cells, and on stellate-shaped cells in the upper dermis at 24 h after injection of PPD; it was maintained up to 1 week. In contrast, no induction of VCAM-1 was seen following challenge with either 2 or 8 MED UV-B. Following PPD, but not UV-B, there was marked induction of ICAM-1 expression on basal keratinocytes. In these biopsies, the inflammation induced in response to PPD therefore differed from UV-B-induced inflammation in showing prolonged expression of endothelial ELAM-1, induction of VCAM-1 on endothelium and other cells, and induction of keratinocyte ICAM-1. These differences may result from differences in the cytokines released and may in turn be responsible for the differences in the nature of the leukocytic infiltration during the two types of inflammatory response.     

Antigen-presenting cells in essential fatty acid-deficient murine epidermis: keratinocytes bearing class II (Ia) antigens may potentiate the accessory cell function of Langerhans cells.

Essential fatty acid deficiency (EFAD) is a useful model for studying the role of (n-6) fatty acid metabolism in normal physiology. Because cutaneous manifestations are among the earliest signs of EFAD and because abnormalities in the distribution and function of tissue macrophages have been documented in EFAD rodents, we studied the distribution and function of Class II MHC (Ia) antigen-bearing cells in EFAD C57B1/6 mouse epidermis. Immunofluorescence studies revealed 1.9-9.6 (mean +/- SEM = 5.2 +/- 2.6) times more class II MHC (Ia) antigen-bearing epidermal cells in suspensions prepared from EFAD as compared to normal skin. Analysis of epidermal sheets demonstrated similar numbers of dendritic Ia+ and NLDC145+ cells in EFAD and normal epidermis, however. This discrepancy occurred because some keratinocytes in EFAD epidermal sheets expressed class II MHC (Ia) antigens, whereas keratinocytes in normal mouse epidermis did not. Two-color flow cytometry confirmed that all Ia+ cells in normal epidermis are Langerhans (Ia+ NLDC145+) cells, whereas Ia+ cells in EFAD epidermis are comprised of Langerhans cells and a subpopulation of keratinocytes (Ia+ NLDC145-). Similar levels of Ia antigens were expressed on EFAD and normal Langerhans cells. EFAD and normal epidermal cells were also compared in several in vitro assays of accessory cell function. Epidermal cells prepared from EFAD C57B1/6 mice present the protein antigen DNP-Ova to primed helper T cells more effectively than epidermal cells prepared from normal animals. EFAD epidermal cells are also more potent stimulators of T cells in primary and secondary allogeneic mixed lymphocyte-epidermal cell reactions than normal epidermal cells. The functional differences between EFAD and normal epidermal cells do not appear to result from increased cytokine release or decreased prostaglandin production by EFAD epidermal cells. In view of these findings and the observation that the antigen-presenting cell activity of EFAD epidermal cells correlates with the number of Ia+ keratinocytes in epidermal cell preparations, Ia+ keratinocytes (in the presence of Langerhans cells) may potentiate cutaneous immune responses in vitro and perhaps in vivo as well. These results also suggest that (n-6) fatty acids or metabolites of (n-6) fatty acids are involved in regulating the expression of class II MHC (Ia) antigens by keratinocytes in vivo.     

Increased expression of Fas on human epidermal cells after in vivo exposure to single-dose ultraviolet (UV) B or long-wave UVA radiation

BACKGROUND: Apoptosis has been proposed to act as an important mechanism for eliminating keratinocytes that have been irreversibly damaged by ultraviolet (UV) irradiation. One way to induce apoptosis in keratinocytes is through activation of the cell surface receptor Fas (CD95), either with the ligand (FasL) or directly with UV radiation. OBJECTIVES: To investigate the regulation of Fas and FasL expression in human skin and the formation of apoptotic cells after in vivo exposure to UVB or long-wave UVA radiation. METHODS: Volunteers were irradiated with either 3 minimal erythema doses (MED) of UVB (n = 6) or 3 MED of long-wave UVA (n = 6) on buttock skin 12, 24 and 72 h before skin punch biopsies were taken. Expression of Fas and FasL was demonstrated by immunohistochemistry on cryostat sections. Apoptosis was assessed by the terminal deoxynucleotidyl transferase-mediated fluorescein-deoxyuridine triphosphate nick-end labelling reaction. RESULTS: In five of six subjects, exposure to UVB radiation resulted in increased homogeneous expression of Fas on epidermal cells, with greatest expression at 24 and 72 h after irradiation. In all subjects, exposure to long-wave UVA resulted in increased homogeneous expression of Fas on epidermal cells, with greatest expression at 12 h after irradiation. In five of six subjects, exposure to UVB radiation resulted in temporarily decreased expression of FasL, but after 72 h the expression of FasL had returned to the preirradiation level. The expression of FasL on epidermal cells after exposure to long-wave UVA showed considerable variation. UVB irradiation was a stronger inducer of epidermal apoptosis than was UVA irradiation. The number of apoptotic epidermal cells did not correlate with expression of Fas or FasL. CONCLUSIONS: In human skin the expression of Fas on epidermal cells increases after in vivo exposure to UVB or long-wave UVA. Exposure to UVB causes a temporary decrease in the expression of FasL on epidermal cells.     

Magnesium ions inhibit the antigen-presenting function of human epidermal Langerhans cells in vivo and in vitro. Involvement of ATPase, HLA-DR, B7 molecules, and cytokines

The combination of seawater baths and solar radiation at the Dead Sea is known as an effective treatment for patients with psoriasis and atopic dermatitis. Dead Sea water is particularly rich in magnesium ions. In this study we wished to determine the effects of magnesium ions on the capacity of human epidermal Langerhans cells to stimulate the proliferation of alloreactive T cells. Twelve subjects were exposed on four subsequent days on the volar aspects of their forearms to 5% MgCl2, 5% NaCl, ultraviolet B (1 minimal erythemal dose), MgCl2 + ultraviolet B, and NaCl + ultraviolet B. Epidermal sheets were prepared from punch biopsies and were stained for ATPase and HLA-DR. Compared with untreated skin, the number of ATPase+/HLA-DR+ Langerhans cells was significantly reduced after treatment with MgCl2 (p = 0.0063) or ultraviolet B (p = 0.0005), but not after NaCl (p = 0.7744). We next questioned whether this reduced expression of ATPase and HLA-DR on Langerhans cells bears a functional relevance. Six subjects were treated on four subsequent days with 5% MgCl2, ultraviolet B (1 minimal erythemal dose), and MgCl2 + ultraviolet B. Epidermal cell suspensions from treated and untreated skin were assessed for their antigen-presenting capacity in a mixed epidermal lymphocyte reaction with allogeneic naive resting T cells as responder cells. Treatment with MgCl2, similarly to ultraviolet B, significantly reduced the capacity of epidermal cells to activate allogeneic T cells (p = 0.0356). Magnesium ions also suppressed Langerhans cells function when added to epidermal cell suspensions in vitro. The reduced antigen-presenting capacity of Langerhans cells after treatment with MgCl2 was associated with a reduced expression by Langerhans cells of HLA-DR and costimulatory B7 molecules, and with a suppression of the constitutive tumor necrosis factor-alpha production by epidermal cells in vitro. These findings demonstrate that magnesium ions specifically inhibit the antigen-presenting capacity of Langerhans cells and may thus contribute to the efficacy of Dead Sea water in the treatment of inflammatory skin diseases.     

Inhibition of intercellular adhesio nmolecule-1 (ICAM-1) expression in ultraviolet B-irradiated human antigen-presenting cells is restored after repair of cyclobutane pyrimidine dimers

The present study assessed the molecular mechanism underlying ultraviolet (UV) B radiation-induced inhibition of the expression of the adhesion molecule ICAM-1 in human antigen-presenting cells (APC). UVB radiation-induced inhibition of ICAM-I expression in human peripheral blood monocytes was associated with the generation of cyclobutane pyrimidine dimers (CPD). CPD were reduced by 60% after treatment with liposomal packed photolyase, an enzyme which removes CPD after absorption of photoreactivating light. Although incomplete, reduction of CPD was associated with complete restoration of ICAM-1 expression at the mRNA and protein level. Neither reduction of CPD level nor restoration of ICAM-1 expression were observed, if monocytes were treated with empty liposomes, or if they were irradiated with photoreactivating light prior to application of photolyase. DNA damage might also induce soluble mediators capable of autocrine inhibition of ICAM-1 expression. UVB irradiation of monocytes did not induce IL-10 production, but resulted in release of prostaglandin (PG) E2. Treatment of unirradiated monocytes with PGE2 completely inhibited ICAM-1 expression, thus mimicking the UVB effect. Inhibition of monocytic PGE2 production by indomethacin, however, did not restore ICAM-1 expression. These results suggest that formation of CPD is necessary and sufficient for UVB radiation-induced inhibition of ICAM-1 expression. In contrast, PGE2 might serve a paracrine role in UVB radiation-induced immunosuppression.     

Interferon-gamma differentially regulates CD80 (B7-1) and CD86 (B7-2/B70) expression on human Langerhans cells

CD80 (B7-1) and CD86 (B7-2/B70) have recently been identified in cultured human Langerhans cells (LCs), although their role and regulatory properties remain unclear. We present our comparison of the expression of the molecules, mRNAs and the function between CD80 and CD86 in human LCs treated by interferon γ (IFN_-γ). We examined the regulatory properties of CD80 and CD86 expression in human LCs pretreated with IFN_-γ. Flow cytometric analysis indicated that the mean fluorescence intensity of CD86 but not CD80 was enhanced. However, the percentage modulation of both CD80 and CD86 positive cells were significantly up-regulated in a dose-dependent manner, after 48-h culturing with IFN_-γ. The regulatory properties of CD80 and CD86 mRNA expressions in human LC were studied using polymerase chain reaction methods. We found that both CD80 and CD86 mRNA of enriched LCs following IFN_-γ pretreatment for 12 h were higher than those without pretreatment. We have demonstrated that the primary allogeneic mixed epidermal cell-lymphocyte reaction induced by human LCs treated by IFN_-γ increased in a dose-dependent manner. There was a 61.5% inhibition by anti-CD86 monoclonal antibody and a 32.5% inhibition by anti-CD80 monoclonal antibody. These data indicate that the CD80 and CD86 expression of human LCs may be differently regulated by IFN_-γ.