Enhanced expression of the high-affinity receptor for IgE (Fc(epsilon)RI) associated with decreased numbers of Langerhans cells in the lesional epidermis of atopic dermatitis

Epidermal Langerhans cells (LCs) and the high-affinity receptor for IgE (Fc(epsilon)RI) on their surface are considered important in the pathogenesis of atopic dermatitis (AD). We investigated the numbers of epidermal LCs and their Fc(epsilon)RI expression in patients with AD and healthy controls. Biopsy specimens taken from lesional skin from 17 patients with AD, non-lesional skin from five patients with AD and normal skin from five healthy individuals were immunohistochemically stained with a monoclonal antibody against CD1a or with either of two monoclonal antibodies against two different epitopes of Fc(epsilon)RI alpha chain. Many dendritic cells were positively stained with anti-CD1a antibody in the epidermis of each skin sample, and fewer cells were stained with anti-Fc(epsilon)RI antibodies. The numbers of epidermal LCs positive for Fc(epsilon)RI were significantly increased in both lesional and non-lesional skin from AD patients compared with those in normal skin, suggesting important roles of Fc(epsilon)RI+LCs in the pathogenesis of the disease. In contrast, the numbers of total epidermal LCs (CD1a-positive) were decreased in AD lesional skin compared with those in non-lesional skin from AD patients and in normal skin from healthy subjects. Together with our finding that the numbers of epidermal LCs were negatively correlated with the clinical severity of the AD lesions, we concluded that epidermal LCs may decrease in some conditions of AD, probably in lesions with severe inflammation.     

Fc epsilon RI on dendritic cells: a receptor, which links IgE mediated allergic reaction and T cell mediated cellular response.

Dendritic cells (DC) are bone marrow derived cells with strong antigen presenting capacity and can induce primary immune responses by activating naive T cells. Cells of this lineage are called as professional antigen presenting cells (APC), because of their primary function as APC. Since the demonstration of IgE bound epidermal Langerhans cells (LC) in atopic dermatitis (AD) patients, a role of IgE bearing DC as a regulator of IgE mediated allergic reactions is emphasized. Indeed, IgE molecules on DC are functional. DC can take up, process and present IgE bound antigens to T cells more efficiently by means of their surface IgE receptor, FcepsilonRI. In addition to its role as an antigen focusing molecule, DC FcepsilonRI may have another function to induce co-stimulatory signals to T cells, by which Th2 type T cell activation is preferentially induced. Thus, this receptor could serve as an amplifying factor for T cell mediated allergic reactions.     

Demonstration of the high-affinity IgE receptor (FcɛRI) on Langerhans cells of oral mucosa

Abstract Langerhans cells in the skin have recently been shown to bind IgE molecules via a high-affinity IgE receptor. Using two specific antibodies, 29C6 and 6F7, against the α-chain of the high-affinity IgE receptor we here demonstrate that Langerhans cells express this receptor in oral mucosa. A specific antibody. Tül, against the low-affinity IgE receptor showed only low expression of this receptor. High-affinity binding for IgE may be important for induction and support of Langerhans cell-dependent transepithelial IgE-mediated allergic reactions and inflammation.     

Demonstration of the high-affinity IgE receptor on human Langerhans cells in normal and diseased skin

Summary Epidermal dendritic cells of normal adult foreskin, and of lesional skin from patients with atopic eczema, stasis eczema and urticaria pigmentosa are shown to be highly reactive with two different monoclonal antibodies (29C6 and 6F7) specific for extracellular domains of the α-chain of the high-affinity IgE receptor. By their distribution pattern, the reactive cells are Langerhans cells. This is confirmed by immunoelectron microscopic demonstration of Birbeck granules in the labelled epidermal cells. Very weak staining is observed on the same cells with an antibody (TÜl) against the low-affinity IgE receptor. Pre-incubation of the sections with IgE partially blocks binding of 6F7 antibody. Langerhans cells, together with dermal mast cells, can therefore bind IgE with high efficiency, and may in this way participate in IgE-mediated cutaneous diseases.     

Serum IgE and Fc epsilon receptor positive lymphocytes in atopic dermatitis--relation to the background of respiratory atopy

The serum IgE level and the population of Fc epsilon R2+ lymphocytes in peripheral blood of 62 patients with atopic dermatitis (AD) were studied. IgE was increased in the majority of the patients with personal or family history of asthma and/or allergic rhinitis (combined AD). On the contrary, only 63.2% of the patients who have solely AD (pure AD) showed less elevation of IgE. There was no correlation between IgE and the severity of dermatitis in pure AD and rhinitis combined AD. However, asthma combined AD showed a significant positive correlation between IgE and the severity of dermatitis. In AD, the population of Fc epsilon R2+ lymphocytes significantly correlated with IgE, suggesting that Fc epsilon R2+ lymphocytes may play a role in the enhanced synthesis of IgE. However, the increase of Fc epsilon R2+ lymphocytes was found only in combined AD, but not in pure AD. These results suggest that elevated IgE and Fc epsilon R2+ lymphocytes reflect combined respiratory atopy rather than AD itself, although there remains the possibility that IgE may influence the formation or exacerbation of dermatitis in asthma combined AD. As far as the participation of IgE is concerned, pure AD and asthma combined AD could be different groups and should be investigated separately.     

Transforming growth factor-beta1 regulates the expression of the high-affinity receptor for IgE on CD34 stem cell-derived CD1a dendritic cells in vitro

It has been reported that monocytes, Langerhans cells (LC) and other dendritic cells (DC) express the high-affinity receptor for IgE (FcepsilonRI) in patients with atopic diseases. These cells may be instrumental in the control of the immune response and the allergic inflammation. In this context, transforming growth factor beta 1 (TGF-beta1) has been highlighted as a key cytokine involved in the mechanisms aimed to orchestrate tolerance and has been suggested as a candidate gene in atopic diseases. In this report, we investigate the putative role of TGF-beta1 in the regulation of FcepsilonRI on cord blood CD34+ stem cell-derived CD1a+ DC (CD34-derived CD1a+ DC). Kinetic experiments show that FcepsilonRI spontaneously appears on the surface of CD1a+ DC, but decreases when exogenous TGF-beta1 is added at high doses (10 ng per mL) or when endogenous TGF-beta1 is neutralized in the culture conditions. In contrast, low-dose TGF-beta1 (0.5 ng per mL) stabilizes surface FcepsilonRI expression on DC. Increasing TGF-beta1 concentrations leads to the generation of LC-like DC showing an augmentation in stimulatory capacity towards allogeneic T cells. In view of these data, a picture emerges that FcepsilonRI+ on DC is finely modified by the TGF-beta1 concentration in the microenvironment and could be of primary relevance in the context of atopic diseases.     

Fc epsilon receptor II/CD23 positive lymphocytes in atopic dermatitis: II. Infiltration of Fc epsilon R II(+) T cells in the skin lesion

Cells expressing Fc receptors for IgE (Fc epsilon R II) were identified in skin from patients with atopic dermatitis (AD), eczematous dermatitis (ED), and in skin from normal nonatopic subjects, with the use of monoclonal antibodies to human lymphocyte Fc epsilon R II, H107, and to lymphoid cell-surface antigens by double immunofluorescence staining. Two to four percent of infiltrating mononuclear cells expressed Fc epsilon R II, and more than half of these cells were T epsilon cells in both acute and chronic AD lesions. Fc epsilon R II(+) T cells (T epsilon cells) bearing CD8 infiltrated preferentially acute lesions, whereas chronic lesions contained either CD8(+) or CD4(+) T epsilon cells, or both. Fc epsilon R II(+) cells rarely were present in ED lesions. There was no significant correlation between % Fc epsilon R II(+) peripheral blood mononuclear cells and the proportion of lesional Fc epsilon R II(+) cells, extent of skin lesions, or serum IgE levels, implying the selective accumulation of Fc epsilon R II(+) cells in the inflammatory infiltrate of AD. These observations suggest that the increased generation of Fc epsilon R II(+) cells in skin lesions, including CD8 (+) T epsilon cells, is involved in the pathogenesis of AD.     

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.     

The distribution of Fc gamma RI, Fc gamma RII and Fc gamma R III on Langerhans' cells and keratinocytes in normal skin

The distribution of Fc-receptors for IgG (FcR) on human epidermal cells (EC) was characterized in situ using monoclonal antibodies (MoAbs) by indirect immunofluorescence staining of cryosections. The results showed heterogeneity of FcR expression on Langerhans' cells (LC) and keratinocytes (KC). The MoAb IV.3 against FcR II (CDw32) gave granular staining of most LC whereas the MoAb 32.2 against FcR I (CD64) occasionally stained a few dendritic cells. 32.2 demonstrated weak granular staining along the outer aspect of KC in stratum spinosum and stratum basale and intense staining of stratum corneum and stratum granulosum. The MoAbs Leu 11b against FcR III (CD16) and B1D6 reacting with a placental FcR with low affinity for IgG gave intense linear membrane staining of KC. Leu 11b produced strongest staining of stratum granulosum and B1D6 the strongest staining of stratum spinosum and basale. The results confirm our previous observations of FcR in situ on LC and KC in normal skin using functional assays and demonstrate that these EC possess different types of low affinity FcR. The data support the contention of an immune function of KC. FcR may be mediators for interaction between KC and LC. The FcR activity in stratum granulosum may have an immune function as a barrier against microorganisms and other antigens.     

Coupling of contact sensitizers to thiol groups is a key event for the activation of monocytes and monocyte-derived dendritic cells

Strong contact sensitizers are able to induce distinct signal transduction mechanisms in antigen-presenting cells by coupling to cell proteins. The predominant target structures of haptens are thought to be thiol and amino groups in cysteine and lysine residues. We studied whether coupling of small reactive chemicals to thiol or amino groups might be responsible for the activation of monocytes and mature monocyte-derived dendritic cells. Human peripheral blood mononuclear cells were stimulated in vitro with subtoxic concentrations of the strong haptens 5-chloro-2-methylisothiazolinone plus 2-methylisothiazolinone and 2, 4, 6-trinitrochlorobenzene, the thiol-reactive reagents N-hydroxymaleimide and N-ethylmaleimide, as well as the amino-reactive compounds sulfosuccinimidyl acetate and 2-iminothiolane. Flow cytometric quantification of tyrosine phosphorylation in CD14+ monocytes showed that 5-chloro-2-methylisothiazolinone plus 2-methylisothiazolinone, 2, 4, 6-trinitrochlorobenzene, N-hydroxymaleimide, and N-ethylmaleimide but not sulfosuccinimidyl acetate and 2-iminothiolane strongly induced this process. Tyrosine phosphorylation induced by 5-chloro-2-methylisothiazolinone plus 2-methylisothiazolinone and 2, 4, 6-trinitrochlorobenzene was completely prevented in the presence of cysteine but not lysine, suggesting a competitive mechanism between cysteine and sulfhydryl groups of cell proteins. Using the mouse ear swelling test N-hydroxymaleimide could be classified as a significant contact allergen in comparison to 2, 4, 6-trinitrochlorobenzene, whereas no sensitizing potential became apparent for sulfosuccinimidyl acetate and 2-iminothiolane. Western blot analysis on monocytes and mature monocyte-derived dendritic cells confirmed the flow cytometric data for tyrosine phosphorylation and demonstrated a selective capacity of haptens and thiol-reactive compounds to activate ERK1/2 mitogen-activated protein kinase. Our data show that strong affinity of a small reactive chemical toward thiol groups is important for the activation of monocytes and monocyte-derived dendritic cells and can support the process of sensitization.     

Wnt5a inhibits the CpG oligodeoxynucleotide-triggered activation of human plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDCs) fulfil multiple roles in immunity, and can secrete large amounts of interferon (IFN)-α. However, the available evidence suggests that they may actually counteract efficient antitumour immunity. Thus in melanoma, pDCs are abundant, but they are anergic and deficient in IFN-α secretion. pDC anergy is thought to be caused by immunosuppressive factors secreted by melanoma cells. One factor strongly expressed by melanoma is Wnt5a, which is implicated in cancer tissue invasion. In this paper, we show that Wnt5a is able to block the upregulation of the activation markers CD80 and CD86 on naive human pDCs stimulated by CpG oligodeoxynucleotide, and CpG-triggered secretion of IFN-α by pDCs. Our results suggest that Wnt5a may not only initiate cancer invasion, but could also regulate activation of pDC. These data provide a clear rationale to investigate a role for Wnt5a in immune regulation.     

Phenotypic and functional outcome of human monocytes or monocyte-derived dendritic cells in a dermal equivalent.

The dermis harbors a true dendritic cell population that could elicit primary allogeneic T cell responses in vitro and contact hypersensitivity reactions in vivo. The origin of dermal dendritic cells remains poorly understood, however. In this study, we analyzed the fate of monocytes or monocyte-derived dendritic cells in a dermal equivalent. Freshly isolated monocytes or monocytes cultured for 6 d with either GM-CSF/IL-4 or GM-CSF/IL-4/TGF-beta 1 (TGF-DC) were seeded in a collagen solution with normal human fibroblasts. The lattices were cultured for 7--14 d in the presence, or absence, of the exogenous cytokines, before phenotypic and functional studies were performed. Supply of exogenous cytokines allows the appearance of typical CD1a(+)/CD14(-)/CD68(low) dendritic cells with significant allostimulatory property, regardless of the cell type incorporated into the lattices. In cytokine-free conditions, monocytes and GM-CSF/IL-4-derived dendritic cells give rise to a CD1a(-)/CD14(+)/CD68(high) monocyte/macrophage population with no allostimulatory property. When incorporated into the lattices in the absence of exogenous cytokines the TGF-DC express few CD68 and FXIIIa. Interestingly, these cells do not all convert into the CD14(+)/CD1a(-) population. Indeed, a small HLA-DR(+)/CD1a(+)/CD14(-) subset was consistently found, which represents about one-third of the HLA-DR(+) cells. Moreover, TGF-DC recovered from the lattices after culture without cytokines do display a significant allostimulatory function. Thus, in the absence of exogenous cytokines, only Langerhans-cell-like dendritic cells can retain the typical dendritic cell features when inserted in a dermal environment. Taken together, these results may provide evidence supporting an epidermal origin of dermal dendritic cells.     

In vivo UVB irradiation induces clustering of Fas (CD95) on human epidermal cells

In vitro studies with human cell lines have demonstrated that the death receptor Fas plays a role in ultraviolet (UV)-induced apoptosis. The purpose of the present study was to investigate the relation between Fas expression and apoptosis as well as clustering of Fas in human epidermis after a single dose of UVB irradiation. Normal healthy individuals were irradiated with three minimal erythema doses (MED) of UVB on forearm or buttock skin. Suction blisters from unirradiated and irradiated skin were raised, and Fas, FasL, and apoptosis of epidermal cells quantified by flow cytometry. Clustering of Fas was from skin biopsied. Soluble FasL in suction blister fluid was quantified by ELISA. Flow cytometric analysis demonstrated increased expression intensity of Fas after irradiation, with 1.6-,2.2- and 2.7-fold increased median expression at 24, 48 and 72 h after irradiation, respectively (n=4). Apoptosis was demonstrated by the TUNEL reaction, and the maximum of apoptotic cells was detected at 48 h after irradiation. Double-staining of Fas and TUNEL showed that apoptosis was restricted to the Fas-positive epidermal subpopulation, but there was no correlation between the intensities of Fas expression and TUNEL reaction. Median expression intensity of FasL-positive cells transiently decreased to 0.9- and 0.8-fold of the preirradiation respective level after 24 h and 48 h, respectively, and returned to the respective preirradiation level at 72 h after irradiation (n=4). Concentrations of soluble FasL in suction blister fluid from UVB-irradiated skin did not differ from those in unirradiated skin (n=5). Confocal laser scanning microscopy showed a rapid clustering of Fas within 30 min after irradiation. A simultaneous clustering of the adapter signalling protein FADD suggested that Fas clustering has a functional significance. Our results ar in accordance with previous findings from in vitro studies, and suggest that Fas is activated in vivo in human epidermis after UVB exposure.     

Interferon‐γ induces upregulation and activation of the interleukin‐31 receptor in human dermal microvascular endothelial cells

Abstract: Interleukin‐31 (IL‐31), a recently discovered cytokine derived from T helper cells, is involved in chronic dermatitis and pruritus. This study demonstrates for the first time that the IL‐31 receptor complex for IL‐31 is substantially upregulated in human dermal microvascular endothelial cells after stimulation with interferon‐γ (IFN‐γ). Activation of the IL‐31 receptor complex results in the induction of the intracellular ERK1/2 signaling pathway and downregulation of IFN‐γ‐induced monokine induced by IFN‐γ expression. Inhibitor studies revealed that the IFN‐γ‐induced IL‐31RA upregulation is processed via JNK and PI3 kinase activation. In sum, our study points toward an interaction between the T_H1‐derived cytokine IFN‐γ and the T_H2‐derived cytokine IL‐31 on endothelial cells.     

Targeting of the hNC16A collagen domain to dendritic cells induces tolerance to human type XVII collagen

Antibodies, specific to murine DEC205, can be used to target antigens to dendritic cells. The immunodominant domain of human type XVII collagen, hNC16A, was fused to this antibody (DEC-hNC16A) and was administered as expression plasmid by gene gun transfection with the aim of inducing tolerance to human type XVII collagen in a skin transplantation model. Mice transfected with DEC-hNC16A were challenged with skin grafts from transgenic mice engineered to express human type XVII collagen. Graft survival was either prolonged or grafts were accepted infinitely (33% and 16%, respectively) upon treatment with DEC-hNC16A while 100% of grafts were rejected in untreated controls. Graft acceptance was associated with the absence of a CD4+ infiltrate and a dense CD8+ T-cell infiltrate and was not strictly dependent on antibody production. Our results show that DEC-hNC16A targets dendritic cells in vivo leading to prolonged survival of transgenic skin grafts. This indicates that DEC205-targeting may be used for the induction of tolerance to skin antigens, which would increase the chances of successful skin gene therapy of epidermolysis bullosa patients.