Reduction of inflammatory slan (6‐sulfo LacNAc) dendritic cells in psoriatic skin of patients treated with etanercept

Dermal dendritic cells (DCs) play a central role in the immunopathology of psoriasis. We previously identified slanDCs as pro‐inflammatory TNF‐α, IL‐23‐ and IL‐12‐producing DCs in human blood and as prominent inflammatory dermal TNF‐α secreting and CD11c‐positive DC subset in psoriasis. Here, we ask for the effects of TNF‐α‐inhibition on inflammatory slanDCs in skin and blood of 10 patients with psoriasis during 24 weeks of treatment with etanercept. Treatment with etanercept reduced the frequency of dermal slanDCs but did not induce apoptosis as determined by lack of increased active caspase‐3‐expression. In parallel, we found increased frequencies of slanDCs in blood which expressed lower levels of HLA‐DR. Stimulating slanDCs isolated from the blood of healthy donors in vitro induced a strong production of IL‐1β, IL‐6, IL‐23 and IL‐12p70. This capacity was efficiently reduced in the presence of etanercept, thereby indicating that TNF‐α is an autocrine stimulus for maturation and pro‐inflammatory cytokine production of slanDCs. In vivo, we noticed that treatment with etanercept did reduce the number of dermal slanDCs in parallel to the overall expression of TNF‐α and IL‐23p19. However, successful treatment did not down‐regulated the percentage of dermal slanDCs that stained positive for TNF‐α and IL‐23p19 indicating that remaining slanDCs kept their pro‐inflammatory capacity. This study provides novel insights into the immune regulatory properties of etanercept at the level of inflammatory slanDCs in vivo in skin and blood as well as in vitro.     

Human 6-sulfo LacNAc (slan) dendritic cells are a major population of dermal dendritic cells in steady state and inflammation

Background. The immune system of the skin has a network of resident dendritic cells (DCs) consisting of epidermal Langerhans cells and various subsets of dermal DCs. We recently reported on a new population of dermal DCs, called slan (6‐sulfoLacNAc+) DCs, which have a potent capacity to stimulate Th17/Th1 T‐cell responses. Aim. To understand the characteristics of slanDCs as a new population of dermal DCs in the context of other DC populations in healthy and psoriatic skin. Methods. We immunofluorescently stained skin samples from healthy controls and from patients with psoriasis. Results. Staining healthy skin for DCs showed that slanDCs (CD1a? CD1c? CD11c? CD14? CD163?) were present at a similar frequency to that of CD1c+ CD11c+ CD1a+ CD14? CD163? dermal DCs, which have previously been regarded as the major population of resident DCs. In psoriatic skin, the frequency of slanDCs and CD1c+ DCs was doubled, and the slanDCs expressed CD11c. In‐depth analysis of DCs in psoriatic skin by four‐colour immunofluorescence analysis showed that the pool of CD11c+ cells could be further subdivided into CD11c+ CD14+ CD163? DCs and CD11c+ CD163+ CD14+ macrophages. Conclusion. SlanDCs, initially described as large population of proinflammatory DCs in blood, are a novel and major part of the resident dermal myeloid DC system in both healthy and inflamed skin.     

Dimethyl‐ and monomethylfumarate regulate indoleamine 2,3‐dioxygenase (IDO) activity in human immune cells

Fumaric acid esters (FAEs) are used as an oral treatment for psoriasis. Dimethylfumarate (DMF) and its metabolite monomethylfumarate (MMF) are regarded as the pharmacologically active moieties. Indoleamine 2,3‐dioxygenase (IDO) is the key enzyme for the metabolism of tryptophan. The kynurenine pathway is established as a major regulator of innate and adaptive immunity. Here, we investigated the effect of DMF and MMF on IDO activity and expression in human peripheral blood mononuclear cells (PBMCs). IDO activity was determined by measuring the concentration of kynurenine in the culture medium using a HPLC technique. IDO and kynureninase protein expressions were analysed by Western blot. Our results demonstrated that DMF and MMF dose‐dependently reduced the levels of L‐kynurenine in PBMCs activated by interferon‐γ (IFN‐γ). Furthermore, MMF had an inhibitory effect on IDO activity in vitro with an ED₅₀ of 10 μmol/L, a value within the therapeutic concentration range for this molecule. We also observed that IDO and kynureninase expressions were reduced in PBMCs in a dose‐dependent manner by DMF and MMF. The results of our study show that DMF and MMF (in therapeutic concentrations) inhibited IDO and kynureninase activity and expression in a NF‐κB‐dependent manner in PBMCs while also decreasing the level of L‐kynurenine in these cells. As we found that FAEs inhibit both IDO expression and enzymatic activity leading to a modulation of tryptophan degradation, we believe this effect may contribute to the clinical efficacy of this drug in psoriasis by downregulating pro‐inflammatory mediators generated by the kynurenine pathway.     

Primary human keratinocytes efficiently induce IL‐1‐dependent IL‐17 in CCR6+ T cells

Abstract: Keratinocytes and activated T cells interact in skin inflammation by virtue of chemokines and cytokines. T cell‐derived IL‐17 has been described to play an important role in the course of psoriatic inflammation. In this study, we addressed how keratinocytes influence the secretion of IL‐17 in autologous T cell subsets. We found that a co‐culture of autologous keratinocytes and T cell‐receptor‐stimulated T cells markedly enhanced the production of IL‐17. Besides the importance of direct cell contact, this effect was mainly mediated by IL‐1 and could be blocked by the IL‐1 antagonist anakinra. An additional increase in IL‐17 production by IL‐23 was only seen in the presence of IL‐1, which thus plays a permissive role for the action of IL‐23. Importantly, co‐culture of keratinocytes with CCR6+ CD4+ T cells that are enriched for Th17 cells resulted in significantly higher IL‐17 production compared to co‐culture with CD4+ T cells.     

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     

In vitro and in vivo evidence of tyrosinase inhibitory activity of a synthesized (Z)‐5‐(3‐hydroxy‐4‐methoxybenzylidene)‐2‐thioxothiazolidin‐4‐one (5‐HMT)

Tyrosinase is a key enzyme that catalyses the initial rate‐limiting steps of melanin synthesis. Due to its critical role in melanogenesis, various attempts were made to find potent tyrosinase inhibitors although many were not safe and effective in vivo. We evaluated tyrosinase inhibitory activity of six compounds. Among them, (Z)‐5‐(3‐hydroxy‐4‐methoxybenzylidene)‐2‐thioxothiazolidin‐4‐one (5‐HMT) had the greatest inhibitory effect and potency as the IC₅₀ value of 5‐HMT was lower than that of kojic acid, widely‐known tyrosinase inhibitor. Based on in silico docking simulation, 5‐HMT had a greater binding affinity than kojic acid with a different binding conformation in the tyrosinase catalytic site. Furthermore, its skin depigmentation effect was confirmed in vivo as 5‐HMT topical treatment significantly reduced UVB‐induced melanogenesis in HRM2 hairless mice. In conclusion, our study demonstrated that 5‐HMT has a greater binding affinity and inhibitory effect on tyrosinase and may be a potential candidate for a therapeutic agent for preventing melanogenesis.     

树突状细胞在炎症性皮肤病中的作用

树突状细胞为皮肤内最重要的抗原提呈细胞，参与免疫反应全过程，其在各类炎症性皮肤病的发病中可能有重要意义。该文对炎症性皮肤病中的树突状细胞亚群，及其表面IgE受体结构和树突状细胞对T细胞亚群的分化迁移作用作一综述。     

Epidermal DR+T6- dendritic cells in inflammatory skin diseases

T lymphocyte and dendritic cell subpopulations were counted in three biopsies each of endogenous eczema and pityriasis rosea and two of lichen planus and compared with previous findings in psoriatic lesions. In common with psoriasis, proportionately more CD4 T cells than CD8 T cells were DR+ in both epidermis and dermis of all lesions. In addition, total numbers of epidermal dendritic cells were significantly increased in endogenous eczema and pityriasis rosea, and variably in lichen planus lesions. Interestingly, a DR+T6- subpopulation of dendritic cells was present in varying proportions in all three skin lesion types. Electron microscopy of DR+T6- dendritic cells from psoriatic lesions, using an immunogold staining technique, showed the cells to be of the Langerhans' cell lineage. DR+T6- dendritic cells are a subpopulation of Langerhans' cells which are not specific to psoriasis, but present in the lesions of other benign, inflammatory skin conditions in which CD4 T cells are preferentially activated.     

The role of dendritic cells during infection

The skin and the mucosa of the respiratory and gastrointestinal tracts are continuously exposed to microorganisms, but only a limited number of these enter the body and cause disease. To resist microbial infection, the host has developed a multitude of defense mechanisms involving the innate and adaptive immune systems. Dendritic cells (DCs) provide the link between these arms of the immune system. The initiation of an immune response is critically dependent on the activation of DCs, which can discriminate between different classes of microorganisms and elicit tailored antimicrobial immune responses. They have an extraordinary capacity to stimulate naive T cells and initiate primary immune responses. In turn, some pathogens interfere with DC function to block or delay their elimination by the host. Progress in understanding the role of DCs in the host response to microbes is reviewed.     

Plasmacytoid dendritic cells: a new cutaneous dendritic cell subset with distinct role in inflammatory skin diseases

Epidermal dendritic cells found in inflamed skin include Langerhans cells and the recently identified population of inflammatory dendritic epidermal cells. Another subset of dendritic cells in humans is the plasmacytoid dendritic cell in peripheral blood, which is characterized by the production of large amounts of type I interferon (interferon-alpha and interferon-beta) upon viral infection. We hypothesized that plasmacytoid dendritic cells might be involved in anti-viral defense mechanisms of the skin. Here we investigated plasmacytoid dendritic cells, inflammatory dendritic epidermal cells, and Langerhans cells in epidermal single cell suspensions of normal looking skin from healthy volunteers and of lesional skin from patients with different inflammatory skin diseases. Langerhans cells were found in normal and in inflamed skin samples. In normal skin, plasmacytoid dendritic cells and inflammatory dendritic epidermal cells were low or absent. Lesional skin samples from patients with psoriasis vulgaris and contact dermatitis contained relatively high numbers of both inflammatory dendritic epidermal cells and plasmacytoid dendritic cells. In contrast, many inflammatory dendritic epidermal cells but only very few plasmacytoid dendritic cells could be detected in atopic dermatitis lesions. Lupus erythematosus was characterized by high numbers of plasmacytoid dendritic cells but low numbers of inflammatory dendritic epidermal cells. These results demonstrate that in addition to resident Langerhans cells, plasmacytoid dendritic cells and inflammatory dendritic epidermal cells are selectively recruited to the skin lesions depending on the type of skin disease. The lack of plasmacytoid dendritic cells in atopic dermatitis may predispose atopic dermatitis patients to viral infections such as eczema herpeticum, a secondary infection of atopic dermatitis lesions with herpes simplex virus. The composition of dendritic cell subsets may help to clarify the etiology of inflammatory skin diseases and forms the basis for therapeutic intervention with selective microbial molecules such as immunostimulatory CpG oligonucleotides.     

Enhanced T‐cell activation by immature dendritic cells loaded with HSP70‐expressing heat‐killed melanoma cells

Please cite this paper as: Enhanced T‐cell activation by immature dendritic cells loaded with HSP70‐expressing heat‐killed melanoma cells. Experimental Dermatology 2010; 19: 108–116. Abstract: Vaccination protocols that utilize dendritic cells (DCs) to elicit therapeutic immunity against tumors are the subject of intense research. Given that the capacity of DCs to cross‐present antigens is physiologically low, there is considerable interest to develop strategies that enhance that pathway. In order to best exploit the enhanced cross‐presentation of antigens bound to heat shock protein 70 (HSP70), we analysed melanoma cell preparations for their HSP70 expression. Western blotting revealed strong upregulation of HSP70 after heat‐killing in contrast to UV‐B irradiation. When the uptake of heat‐killed necrotic cells by DCs at various levels of maturation was assessed, 61 ± 7% of immature DCs (iDCs) internalized fluorescence‐labelled necrotic material. Apoptotic material from UV‐B‐irradiated cells was internalized by only 48 ± 5% of iDCs. Maturation‐inducing cytokines did not affect the uptake when added simultaneously with the tumor cell preparations. Loading DCs with heat‐necrotic or apoptotic melanoma cells slightly reduced CD83 expression while leaving CD208 (DC‐LAMP) expression unchanged. As determined by IFN‐γ‐detecting enzyme‐linked‐immunospot assays, iDCs loaded with heat‐killed melanoma cells activated autologous T cells most effectively when used without any further maturation, whereas DCs loaded with apoptotic material required maturation. In conclusion, HSP70‐expressing melanoma cells could be generated by heat‐killing. Loading iDCs with heat‐killed melanoma cells resulted in a superior priming of autologous T cells in vitro.     

Interleukin (IL)‐6 modulates transforming growth factor‐β receptor I and II (TGF‐βRI and II) function in epidermal keratinocytes

It been shown that IL‐6 modulates TGF‐β1 expression in fibroblasts, however, what role IL‐6 plays concerning TGF‐βR expression and function in skin is unknown. Therefore, the aim of this study was to investigate the mechanism by which IL‐6 might modulates TGF‐β receptors in skin. Skin from WT, IL‐6 over‐expressing mice and IL‐6 treated keratinocyte cultures was analysed for TGF‐βRI and TGF‐βRII expression via histology, PCR and flow cytometry. Receptor function was assessed by cell migration, bromodeoxyuridine (BrdU) proliferation assays, and Smad7 expression and Smad2/3 phosphorylation. Receptor localization within the membrane was determined by co‐immunoprecipitation. IL‐6 overexpression and treatment increased TGF‐βRII expression in the epidermis. IL‐6 treatment of keratinocytes induced TGF‐βRI and II expression and augmented TGF‐β1‐induced function as demonstrated through increased migration and decreased proliferation. Additionally, IL‐6 treatment of keratinocytes altered receptor activity as indicated by altered Smad2/3 phosphorylation and increased Smad7 and membrane localization. These results suggest that IL‐6 regulates keratinocyte function by modulating TGF‐βRI and II expression and signal transduction via trafficking of the receptor to lipid raft pools.     

Update on the role of plasmacytoid dendritic cells in inflammatory/autoimmune skin diseases

Plasmacytoid dendritic cells (pDCs) represent a specialized dendritic cell population that exhibit plasma cell morphology, express CD4, CD123, blood‐derived dendritic cell antigen‐2 (BDCA‐2) and Toll‐like receptor (TLR)7 and TLR9 within endosomal compartments. When activated, pDCs are capable of producing large quantities of type I IFNs (mainly IFN‐α/β), which provide antiviral resistance and link the innate and adaptive immunity. While generally lacking from normal skin, pDCs infiltrate the skin and appear to be involved in the pathogenesis of several inflammatory, infectious (especially viral) and neoplastic entities. In recent years, pDC role in inflammatory/autoimmune skin conditions has been extensively studied. Unlike type I IFN‐mediated protective immunity that pDCs provide at the level of the skin by regulated sensing of microbial or self‐nucleic acids upon skin damage, excessive sensing may elicit IFN‐driven inflammatory/autoimmune diseases. In this review, focus will be on the role of pDCs in cutaneous inflammatory/autoimmune dermatoses.     

Internalization routes of cell‐penetrating melanoma antigen peptides into human dendritic cells

Optimized delivery of antigens combined with sustainable maturation of dendritic cells (DCs) is crucial for generation of effective antitumoral immune responses. Multiple approaches for ex vivo antigen loading and improvement in immunogenicity have been described. We have recently established a single‐step protocol consisting of a fusion peptide (a sequence of the melanoma antigen Melan‐A and a cationic cell‐penetrating HIV TAT domain) bound in complexes with a toll‐like receptor agonist. As the exact cellular uptake mechanisms of TAT‐coupled antigens have been a matter of considerable debate and significantly depend on cell type, cargo and concentrations, we evaluated internalization routes into human immature DCs in comparison with non‐phagocytic cell lines. We found that Melan‐A‐TAT fusion peptide uptake by DCs is mainly energy dependent, superior compared with polylysine‐coupled Melan‐A and significantly higher in DCs as compared with Jurkat cells or HUVECs. Furthermore, we could track the uptake of the fusion peptide exclusively through early endosomes to lysosome compartments after 90 min by fluorescence microscopy and immunoelectron microscopy. Specific endocytosis inhibitors revealed major internalization of the fusion peptide by DCs via clathrin‐mediated endocytosis, whereas uptake by non‐phagocytic HUVECs differed significantly, involving macropinocytosis as well as clathrin‐mediated endocytosis. As our understanding of the processes involved in internalization of TAT‐coupled cargos by human DCs broadens, and DC activation becomes available by single‐step procedures as described, further development of simultaneous DC maturation and intra‐cellular peptide targeting is warranted.