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Secretomes from various cell sources exert strong regenerative activities on numerous organs, including the skin. Although secretomes consist of many diverse components, a growing body of evidence suggests that small extracellular vesicles (EVs) account for their regenerative capacity. We previously demonstrated that the secretome of γ-irradiated peripheral blood mononuclear cells (PBMCs) exhibits wound healing capacity. Therefore, we sought to dissect the molecular composition of EVs present in the secretome and compared wound healing-related activities of these EVs to other subfractions of the secretome and the fully supplemented secretome (MNCaposec). Compared to EVs derived from non-irradiated PBMCs, γ-irradiation significantly increased the size and number and changed the composition of released EVs. Detailed characterization of the molecular components of EVs, i.e. miRNA, proteins, and lipids, derived from irradiated PBMCs revealed a strong association with regenerative processes. Reporter gene assays and aortic ring sprouting assays revealed diminished activity of the subfractions compared to MNCaposec. In addition, we showed that MNCaposec accelerated wound closure in a diabetic mouse model. Taken together, our results suggest that secretome-based wound healing represents a promising new therapeutic avenue, and strongly recommend using the complete secretome instead of purified subfractions, such as EVs, to exploit its full regenerative capacity.

RTN1 is an endoplasmic reticulum-associated protein that was initially identified in neuronal tissues. Here we show that the main isoform RTN1A is a marker for dendritic cells. In the skin, HLA-DR+CD1ahighCD207+CD11cweak Langerhans cells were the only cells in the epidermis, and HLA-DR+CD11c+ dendritic cells were the main cells in the dermis, expressing this protein. RTN1A+ dendritic cells were also found in gingiva, trachea, tonsil, thymus, and peripheral blood. During differentiation of MUTZ-3 cells into Langerhans cells, expression of RTN1A mRNA and protein preceded established Langerhans cell markers CD1a and CD207, and RTN1A protein partially co-localized with the endoplasmic reticulum marker protein disulfide isomerase. In line with this observation, we found that RTN1A was expressed by around 80% of Langerhans cell precursors in human embryonic skin. Our findings show that RTN1A is a marker for cells of the dendritic lineage, including Langerhans cells and dermal dendritic cells. This unexpected finding will serve as a starting point for the elucidation of the, until now, elusive functional roles of RTN1A in both the immune and the nervous system.

Background CARD18 contains a caspase recruitment domain (CARD) via which it binds to caspase-1 and thereby inhibits caspase-1-mediated activation of the pro-inflammatory cytokine interleukin (IL)-1β. Objectives To determine the expression profile and the role of CARD18 during differentiation of keratinocytes and to compare the expression of CARD18 in normal skin and in inflammatory skin diseases. Methods Human keratinocytes were induced to differentiate in monolayer and in 3D skin equivalent cultures. In some experiments, CARD18-specific siRNAs were used to knock down expression of CARD18. CARD18 mRNA levels were determined by quantitative real-time PCR, and CARD18 protein was detected by Western blot and immunofluorescence analyses. In situ expression was analyzed in skin biopsies obtained from healthy donors and patients with psoriasis and lichen planus. Results CARD18 mRNA was expressed in the epidermis at more than 100-fold higher levels than in any other human tissue. Within the epidermis, CARD18 was specifically expressed in the granular layer. In vitro CARD18 was strongly upregulated at both mRNA and protein levels in keratinocytes undergoing terminal differentiation. In skin equivalent cultures the expression of CARD18 was efficiently suppressed by siRNAs without impairing stratum corneum formation. Epidermal expression of CARD18 was increased after ultraviolet (UV)B irradiation of skin explants. In skin biopsies of patients with psoriasis no consistent regulation of CARD18 expression was observed, however, in lesional epidermis of patients with lichen planus, CARD18 expression was either greatly diminished or entirely absent whereas in non-lesional areas expression was comparable to normal skin. Conclusions Our results identify CARD18 as a differentiation-associated keratinocyte protein that is altered in abundance by UV stress. Its downregulation in lichen planus indicates a potential role in inflammatory reactions of the epidermis in this disease.

Cerebellar degeneration-related antigen 1 (CDR1) was described to be expressed in the nervous system and in different types of cancer tissues. In the present study, we demonstrate that CDR1 is in addition ubiquitously expressed in human epidermis, dermis and isolated skin cells. Both CDR1 mRNA and protein were detected in human skin-derived mast cells, melanocytes, fibroblasts and keratinocytes, suggesting that CDR1 does not have a neuron-specific function.

The advent of organotypic skin models advanced the understanding of complex mechanisms of keratinocyte differentiation. However, these models are limited by both availability of primary keratinocytes and donor variability. Keratinocytes derived from cultured hair follicles and interfollicular epidermis were immortalized by ectopic expression of SV40 and hTERT. The generated keratinocyte cell lines differentiated into stratified epidermis with well-defined stratum granulosum and stratum corneum in organotypic human skin models. They behaved comparable to primary keratinocytes regarding the expression of differentiation-associated proteins, cell junction components and proteins associated with cornification and formed a barrier against biotin diffusion. Mechanistically, we found that SV40 large T-antigen expression, accompanied by a strong p53 accumulation, was only detectable in the basal layer of the in vitro reconstructed epidermis. Inhibition of DNA-methylation resulted in expression of SV40 large T-antigen also in the suprabasal epidermal layers and led to incomplete differentiation of keratinocyte cell lines. Our study demonstrates the generation of keratinocyte cell lines which are able to fully differentiate in an organotypic skin model. Since hair follicles, as source for keratinocytes, can be obtained by minimally invasive procedures, our approach enables the generation of cell lines also from individuals not available for skin biopsies.

Fish oil rich in docosahexaenoic acid (DHA) has beneficial effects on human health. Omega-3 polyunsaturated fatty acids are precursors of eicosanoids and docosanoids, signaling molecules that control inflammation and immunity, and their dietary uptake improves a range of disorders including cardiovascular diseases, ulcerative colitis, rheumatoid arthritis, and psoriasis. The unsaturated nature of these fatty acids, however, makes them prone to oxidation, especially when they are incorporated into (membrane) phospholipids. The skin is an organ strongly exposed to oxidative stress, mainly due to solar ultraviolet radiation. Thus, increased levels of PUFA in combination with oxidative stress could cause increased local generation of oxidized lipids, whose action spectrum reaches from signaling molecules to reactive carbonyl compounds that can crosslink biomolecules. Here, we investigated whether PUFA supplements to fibroblasts are incorporated into membrane phospholipids and whether an increase of PUFA within phospholipids affects the responses of the cells to UV exposure. The redox-sensitive transcription factor Nrf2 is the major regulator of the fibroblast stress response to ultraviolet radiation or exposure to oxidized lipids. Here we addressed how Nrf2 signaling would be affected in PUFA-supplemented human dermal fibroblasts and mouse dermal fibroblasts from Nrf2-deficient and wild type mice. We found, using HPLC-tandem MS, that DHA supplements to culture media of human and murine fibroblasts were readily incorporated into phospholipids and that subsequent irradiation of the supplemented cells with UVA resulted in an increase in 1-palmitoyl-2-(epoxyisoprostane-E2)-sn-glycero-3-phosphorylcholine and Oxo-DHA esterified to phospholipid, both of which are Nrf2 agonists. Also, induction of Nrf2 target genes was enhanced in the DHA-supplemented fibroblasts after UVA irradiation. In Nrf2-deficient murine fibroblasts, the expression of the target genes was, as expected, decreased, but surprisingly, expression of TNFα and MMP13 was strongly induced in DHA-supplemented, UVA-irradiated cells. Also, Nrf2-deficient cells had increased levels of oxidized phospholipids relative to the unoxidized precursors after UVA irradiation. Our data suggest that under ultraviolet stress a functioning Nrf2 system is required to prevent DHA-induced inflammation and matrix degradation in dermal fibroblasts.

Background The human lung is considered a nonsterile organ, and surgical interventions therefore take place in a more or less contaminated operating field. Nevertheless, infectious complications of the pleural cavity are low after major lung resections. Antimicrobial peptides (AMPs) are part of the innate immunity and display a broad capacity to kill pathogens. We hypothesized that the pleural space must have a high natural antimicrobial barrier and that AMPs might effectively protect the pleural cavity. Methods Pleural effusions were collected after lung operations. Antimicrobial activity of the fluids against gram-positive and gram-negative pathogens was analyzed by microdilution assays. AMPs were determined by enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and immunohistochemical analysis. The impact of proinflammatory triggers on AMP release from pleural mesothelial cells was evaluated. Results Antimicrobial activity assays revealed high bactericidal properties of postoperative pleural drainage fluids. They effectively killed gram-negative pathogens ( Escherichia coli , Pseudomonas aeruginosa ) as well as gram-positive pathogens ( Staphylococcus aureus , Streptococcus pneumoniae, and Streptococcus pyogenes ). A variety of AMPs was detected at constantly high concentrations in the pleural fluids. They mainly derived from leukocytes and pleural epithelium. Although proinflammatory cytokine levels were elevated in the postoperative pleural fluids, AMP expression could not be augmented by Toll-like receptor (TLR) triggering or by the proinflammatory cytokines interleukin (IL)-1β and tumor necrosis factor (TNF)α. Conclusions We provide the first evidence of a high abundance of AMPs in postoperative pleural fluids. Our findings might explain the broad protection against infectious complications of the pleural space after major lung operations.

Antimicrobial peptides (AMPs), an important part of the innate immune system, are crucial for defense against invading microorganisms. Whereas AMPs have been extensively studied in adult skin, little is known about the impact of AMPs in the developing human skin. We therefore compared the expression and regulation of AMPs in fetal, neonatal, and adult keratinocytes (KCs) in vitro. The constitutive expression of human β-defensin-2 (HBD-2), HBD-3, S100 protein family members, and cathelicidin was significantly higher in KCs from fetal skin than in KCs from postnatal skin. The capacity to further increase AMP production was comparable between prenatal and postnatal KCs. Analysis of skin equivalents (SEs) revealed a strong constitutive expression of S100 proteins in fetal but not in neonatal and adult SEs. The elevated AMP levels correlated with reduced H3K27me3 (tri-methyl-lysine 27 on histone H3) levels and increased expression of the histone demethylase JMJD3. Knockdown of JMJD3 in fetal KCs elevated H3K27me3 levels and significantly downregulated the expression of HBD-3, S100A7, S100A8, S100A9, and cathelicidin. Our data indicate a crucial contribution of histone modifications in the regulation of AMP expression in the skin during ontogeny. The elevated AMP expression in prenatal skin might represent an essential defense strategy of the unborn.

Cornifelin (CNFN) has been identified as a protein component of epidermal corneocytes. Here, we investigated the tissue distribution of CNFN and potential consequences of CNFN deficiency on epithelial function in in vitro models of human skin and oral mucosa. Our detailed bioinformatics and immunostaining analysis revealed that CNFN is not only expressed in human epidermis but also in noncornifying oral mucosa. In normal epidermis, CNFN was confined to the upper granular layer and the stratum corneum. By contrast, in both partly cornifying and noncornifying oral mucosa, CNFN was expressed in a cell membrane-associated pattern over several suprabasal layers. Small interfering RNA-mediated knockdown of CNFN in epidermal keratinocytes (KCs) was associated with only subtle alterations of the overall epidermal architecture in skin models in vitro but led to altered morphology of corneodesmosomes, as detected by electron microscopy. Using dispase treatment followed by mechanical stress, epithelial sheets of CNFN-deficient epidermal KCs were easily disrupted, whereas their CNFN-competent counterparts remained intact. In contrast to the epidermal KCs, CNFN knockdown in oral KCs had a more severe effect and caused pronounced acantholysis in organotypic models of oral mucosa. Together, these findings indicate that CNFN is a structural component of the cell adhesion system of differentiated KCs in both epidermis and oral mucosa.

Background Neutrophils and allergen-specific T cells accumulate in patients with allergic late-phase reactions (LPRs). Their presence is associated with severe inflammation. Cytokines, such as GM-CSF, IFN-γ, and IL-3, which are typically found in patients with allergic LPRs, have been proposed to convert neutrophils into antigen-presenting cells (APCs). Objective We sought to assess the antigen-processing and antigen-presenting capacities of neutrophils from allergic patients. Methods Neutrophils were isolated from peripheral blood of donors with birch pollen allergy and stimulated with GM-CSF, IFN-γ, and IL-3. The viability and expression of HLA-DR, CD80, and CD86 were assessed by using flow cytometry. HLA-DM expression was analyzed by means of immunoblotting. Allergen uptake was studied after fluorescence labeling of the major birch pollen allergen Bet v 1. Bet v 1 was digested with neutrophilic endolysosomal extracts, and the resulting fragments were sequenced by using mass spectrometry. Neutrophils were used as APCs in coculture experiments with autologous HLA-DR–restricted and Bet v 1–specific T-cell clones reactive with epitopes in different regions of the allergen. In all experiments monocytes were used for comparison. Fluids from suction blisters formed on top of LPRs induced by using intradermal allergen injection were assessed for HLA-DR+ neutrophils by using flow cytometry. Results The cytokines significantly enhanced the survival, allergen uptake, and expression of HLA-DM and HLA-DR on neutrophils. Neutrophils rapidly degraded Bet v 1 into fragments containing all relevant T-cell epitopes. Cytokine-activated, allergen-pulsed neutrophils induced proliferative and cytokine responses of Bet v 1–specific T cells irrespective of epitope specificity, confirming that they fully processed and presented the allergen. HLA-DR+ neutrophils were detected in patients with cutaneous allergic LPRs. Conclusion Neutrophils can serve as APCs for local allergen-specific effector T cells in patients with allergic LPRs.

Background and Purpose Since the identification of the histamine H4 receptor, several ligands activating this receptor have been described and more compounds are in development. These ligands are well characterized in pharmacological assays, including radioligand competition binding studies, GTPγS and GTPase assays. In most cases, these experiments are performed in transfected cell lines, expressing unnaturally high levels of target receptors and G-protein signalling components. In this study we investigated the specific properties of H4 receptor ligands in native cells. Experimental Approach Histamine and five different H4 receptor agonists – 4-methylhistamine, UR-PI376, clobenpropit, VUF8430 and ST-1006 – were characterized in freshly isolated human monocytes. The ligands (10 nM–10 μM) were tested as inhibitors of IL-12p70 secretion from human monocytes and the effects of the H2 receptor antagonist ranitidine and the H4 receptor antagonist JNJ7777120 on their action was investigated. Key Results Histamine and all the tested agonists reduced IL-12p70 secretion into monocyte supernatants by 40–70%. The potencies varied with pEC50 values ranging from 5.7 to 6.9, depending on the agonist used. All potencies were lower than those determined in the original investigations of the compounds. Pretreatment of monocytes with H2 or H4 receptor antagonists showed that some H4 receptor ligands also had low activity at the H2 receptor. Conclusions and Implications Our study demonstrates discrepancies between the potencies obtained from assays in transfected cell lines and assays in native human cells, indicating the importance of evaluating H4 receptor ligands in native cells. Linked Articles This article is part of a themed issue on Histamine Pharmacology Update. To view the other articles in this issue visit http://dx.doi.org/ 10.1111/bph.2013.170.issue-1

Systemic antagonists of the histamine type 1 and 2 receptors (H1/2r) are widely used as anti-pruritics and central sedatives, but demonstrate only modest anti-inflammatory activity. Because many inflammatory dermatoses result from defects in cutaneous barrier function, and because keratinocytes express both Hr1 and Hr2, we hypothesized that H1/2r antagonists might be more effective if they were used topically to treat inflammatory dermatoses. Topical H1/2r antagonists additively enhanced permeability barrier homeostasis in normal mouse skin by the following mechanisms: (i) stimulation of epidermal differentiation, leading to thickened cornified envelopes; and (ii) enhanced epidermal lipid synthesis and secretion. As barrier homeostasis was enhanced to a comparable extent in mast cell-deficient mice, with no further improvement following application of topical H1/2r antagonists, H1/2r antagonists likely oppose mast cell-derived histamines. In four immunologically diverse, murine disease models, characterized by either inflammation alone (acute irritant contact dermatitis, acute allergic contact dermatitis) or by prominent barrier abnormalities (subacute allergic contact dermatitis, atopic dermatitis), topical H1/2r agonists aggravated, whereas H1/2r antagonists improved, inflammation and/or barrier function. The apparent ability of topical H1r/2r antagonists to target epidermal H1/2r could translate into increased efficacy in the treatment of inflammatory dermatoses, likely due to decreased inflammation and enhanced barrier function. These results could shift current paradigms of antihistamine utilization from a predominantly systemic to a topical approach.

Background The function of skin mast cells has been well documented in IgE-mediated allergic reactions, whereas other mast cell functions are poorly defined. This study aimed at identifying novel mast cell proteins by proteome analysis of primary human skin mast cells. Methods The proteome of skin mast cells was compared to other cell types and analyzed using bioinformatics. The expression and function of two proteins hitherto not described in skin mast cells was investigated in isolated mast cells as well as in mast cells in situ. Results Within the mast cell proteome, we identified 49 highly expressed proteins previously not described in mast cells; 21 of these proteins were found to be selectively expressed in mast cells. Two proteins, the neural cell adhesion molecule L1 and dipeptidyl peptidase 4, were further studied. L1 was found to be highly expressed in mast cells in normal, psoriasis, and mastocytosis skin. Dipeptidyl peptidase 4 was found to be expressed in mast cells in normal, psoriasis, and mastocytosis skin as well as in bone marrow mast cells in patients with systemic mastocytosis. In normal skin, mast cells were identified as a major source of dipeptidyl peptidase 4 and we also found that skin mast cells and fibroblasts secrete an active form of this enzyme. Conclusions In a systematic proteomics approach we identified two novel mast cell proteins potentially relevant to skin homeostasis: neural cell adhesion molecule L1 and dipeptidyl peptidase 4.

In normal skin, tightly connected keratinocytes in the stratum granulosum and terminally differentiated keratinocytes in the stratum corneum build an efficient barrier that inhibits extensive water loss while simultaneously preventing the entry of microbial pathogens and allergens into the skin (1). Dysregulation of keratinocyte differentiation together with pathologic changes in this natural barrier function is commonly associated with skin diseases such as atopic dermatitis, psoriasis, and ichthyosis vulgaris (2–4). Recently, loss-of-function mutations in the filaggrin gene have been identified (5, 6). These mutations not only impair the normal formation of the skin barrier, but also decrease the production of endogenous moisturizing molecules that lead to reduced stratum corneum hydration (4, 7). However, besides genetic defects of filaggrin production, other factors play a role in the pathogenesis of atopic dermatitis and the associated skin barrier defects as indicated by several observations: (i) The majority of individuals with atopic dermatitis do not exhibit null mutations in the filaggrin gene (8), (ii) individuals with atopic dermatitis without mutations in the filaggrin gene may also develop severe skin barrier defects (9), and (iii) a significant fraction of individuals carrying double-allele loss-of-function mutations in the filaggrin gene do not develop atopic dermatitis (4, 10). In line with this concept, it was shown recently that the pro-inflammatory cytokines interleukin-4 (IL-4), IL-31, and TNF-α compromise barrier function (11) and modulate the expression of differentiation-associated proteins in keratinocytes (12–14). Mast cells are present in normal skin, and increased numbers of mast cells are regularly observed in the skin of patients with atopic dermatitis even before the onset of inflammation (4, 15). Mast cells release a number of important signaling molecules, among which histamine has particularly potent pro-inflammatory activities (16). After mast cell degranulation, histamine concentrations within the tissue can rise to 10–1000 μM (17), and increased histamine levels have been reported for lesional and nonlesional skin of patients with atopic dermatitis (18). A role of endogenous histamine in the modulation of keratinocyte maturation has been suggested previously based on the observation that antihistamines have a beneficial effect on skin barrier recovery after tape striping in normal mouse skin (19). In the present study, we address the impact of histamine on the differentiation of human keratinocytes in different in vitro systems, among them a three-dimensional organotypic human skin model. This skin model has been shown previously to resemble native human skin, especially with regard to skin development and keratinocyte differentiation (20, 21). Our findings show that histamine prevents the expression of late differentiation antigens in keratinocytes and strongly decreases the expression of tight junction and desmosomal proteins, leading to the formation of a defective skin barrier.

The histamine H4 receptor is functionally expressed on CD4(+) T cells and in particular on human CD4(+) Th2-polarized T cells. Interleukin (IL)-17-producing T cells (Th17 cells) represent a newly defined major CD4(+) T-cell subset, having been identified in psoriatic plaques and in acute skin lesions of atopic dermatitis where histamine is also present in high concentrations. To elucidate the role of the histamine H4 receptor (H4R) on these effector T cells, we polarized human memory T cells into Th17 cells. Further, we investigated H4R expression and assessed its function by real-time PCR, by a cytokine secretion assay of IL-17, and by electrophoretic mobility shift assay of activating protein-1 (AP-1). We show that Th17 cells polarized by IL-1β together with IL-23 express the H4R on mRNA and protein level. Additionally, we identified IL-17-positive cells in psoriatic skin lesions. The IL-17-positive lymphocytes were all positive also for functional H4R. Stimulation with histamine or a H4R agonist increased the production of IL-17 and induced activating protein-1 in Th17 cells. In inflammatory skin diseases with enhanced histamine release, such as psoriasis and atopic dermatitis, histamine might foster the immunomodulatory potency of skin-infiltrating Th17 cells.

The pathology of atopic dermatitis is orchestrated on the cellular level by several different cell types in the characteristic skin lesions. In such lesions, histamine as a mediator of many biological functions is also present in high concentrations. Most of the cells involved in the inflammatory responses express the histamine H1 and H2 receptors, but drugs targeting these receptors are not clinically effective. The discovery of the fourth histamine receptor, which is differentially expressed on immune and nonimmune cells, has shed new light on the actions of histamine in the complexity of atopic dermatitis. In this review, we describe a possible genetic impact on the expression level of the histamine H4 receptor and summarize the current data regarding the activity of the histamine H4 receptor on the key effector cells in atopic dermatitis. We do so in the context of whether the histamine H4 receptor offers a novel target for effective treatments of inflammatory skin diseases.

6-Sulpho LacNAc dendritic cells (slanDC) are a major population of human blood DC that are highly pro-inflammatory, as characterized by their outstanding capacity to produce tumour necrosis factor-α and interleukin-12 (IL-12) and to prime antigen-specific T-cell responses. SlanDC were found to be present in inflamed tissue such as atopic dermatitis, where high levels of histamine are also present. As histamine is an important regulator of allergic inflammation we investigated the role of histamine receptors, particularly the most recently identified histamine H(4) receptor (H(4) R), in modulating the pro-inflammatory function of slanDC. The expression of H(4) R was evaluated by real-time PCR and flow cytometry. Cytokine production in response to H(4) R stimulation was assessed by intracellular flow cytometric staining and enzyme-linked immunosorbent assay. We show that slanDC express the H(1) R, H(2) R and H(4) R on mRNA and the H(4) R on protein level. No differences were observed in basal H(4) R expression in patients with atopic dermatitis and psoriasis, but in atopic dermatitis patients the H(4) R was up-regulated by interferon-γ. When stimulated with lipopolysaccharide in the presence of histamine, slanDC produced substantially lower levels of the pro-inflammatory cytokines tumour necrosis factor-α and IL-12, mediated solely via the H(4) R and via the combined action of H(2) R and H(4) R, respectively. In contrast, the production of IL-10 was not affected by histamine receptor activation on slanDC. The slanDC express the H(4) R and its stimulation leads to reduced pro-inflammatory capacity of slanDC. Hence, H(4) R agonists might have therapeutic potential to down-regulate immune reactions, e.g. in allergic inflammatory skin diseases.

To cite this article: Gschwandtner M, Rossbach K, Dijkstra D, Baumer W, Kietzmann M, Stark H, Werfel T, Gutzmer R. Murine and human Langerhans cells express a functional histamine H4 receptor: modulation of cell migration and function. Allergy 2010; 65: 840–849. Abstract Background: Histamine is an important mediator of allergic reactions, and recent studies indicated that the function of different types of antigen presenting cells (APC) can be modulated by histamine, in particular via the newly described histamine H4 receptor (H4R). Therefore, we investigated possible interactions of histamine via the H4R on Langerhans cells (LC), which represent the professional APC in the skin and therefore have an important role in the initiation and maintenance of allergic skin diseases. Methods: The expression of the H4R was evaluated by real-time PCR, flow cytometry and immunofluorescence staining. The function of the H4R was determined by intracellular flow cytometric measurement of chemokine production and LC migration assays. Results: Here, we show H4R expression on in vitro generated monocyte-derived LC (mRNA and protein) and on primary LC from murine and human skin samples (protein). The immunofluorescence staining in murine and human skin samples clearly proved that LC express the H4R in situ. Stimulation with histamine or a H4R agonist downregulated the chemokine (C-C motif) ligand 2 (CCL2) in human monocyte-derived LC and primary LC. Prestimulation with a selective H4R antagonist abolished this effect. Moreover, migration of LC from the epidermis was increased after H4R agonist stimulation in ex vivo migration assays using human epidermis and murine in vivo assays. Conclusion: Our findings show that LC express a functional H4R and point towards a possible pathogenic relevance of the H4R in inflammatory and allergic diseases.

Skin inflammation and the migration of cells at the site of the immune response play an important role in allergic skin diseases. It has already been described that matrix metalloproteinase 9 (MMP-9) influences tissue remodeling and facilitates cell migration by proteolytic degradation of basal membrane components. The aim of this study was to investigate MMP-9 expression on human primary keratinocytes (KCs) upon stimulation with histamine, a potent mediator in allergic responses. With ELISA and zymography, we could show that histamine induced dose-dependent upregulation of MMP-9 in cultured KCs and in punch biopsies of human skin. The histamine H(1) receptor (H(1)R) agonist beta-histine-but not agonists for H(2)R, H(3)R, and H(4)R-induced MMP-9, whereas the H(1)R antagonist clemastine blocked the effect in a dose-dependent manner. Immunohistological staining showed that histamine-induced MMP-9 led to destruction of type IV collagen at the basement membrane in healthy skin. In a coculture system of KCs and T cells, migration of T cells through an artificial basement membrane was increased after histamine stimulation of KCs. Our findings demonstrate enhanced MMP-9 production and cell migration after histamine stimulation and may represent a new mechanism by which KCs contribute to the pathology of skin diseases.

Reverse transcription polymerase chain reaction (qRT-PCR) has become a mainstay in many areas of skin research. To enable quantitative analysis, it is necessary to analyse expression of reference genes (RGs) for normalization of target gene expression. The selection of reliable RGs therefore has an important impact on the experimental outcome. In this study, we aimed to identify and validate the best suited RGs for qRT-PCR in human primary keratinocytes (KCs) over a broad range of experimental conditions using the novel bioinformatics tool 'RefGenes', which is based on a manually curated database of published microarray data. Expression of 6 RGs identified by RefGenes software and 12 commonly used RGs were validated by qRT-PCR. We assessed whether these 18 markers fulfilled the requirements for a valid RG by the comprehensive ranking of four bioinformatics tools and the coefficient of variation (CV). In an overall ranking, we found GUSB to be the most stably expressed RG, whereas the expression values of the commonly used RGs, GAPDH and B2M were significantly affected by varying experimental conditions. Our results identify RefGenes as a powerful tool for the identification of valid RGs and suggest GUSB as the most reliable RG for KCs.

Background Epidermal hyperproliferation resulting in acanthosis is an important clinical observation in patients with atopic dermatitis, and its underlying mechanisms are not completely understood. Objective Because increased levels of histamine are present in lesional skin, we investigated the effect of histamine, especially with regard to histamine 4 receptor (H4R) activation, on the proliferation of human and murine keratinocytes. Methods The expression of H4R on human and murine keratinocytes was detected by using real-time PCR. Keratinocyte proliferation was evaluated by using different in vitro cell proliferation assays, scratch assays, and measurement of the epidermal thickness of murine skin. Results We detected H4R mRNA on foreskin keratinocytes and on outer root sheath keratinocytes; H4R mRNA was more abundant in keratinocytes from patients with atopic dermatitis compared with those from nonatopic donors. Stimulation of foreskin keratinocytes, atopic dermatitis outer root sheath keratinocytes, and H4R-transfected HaCaT cells with histamine and H4R agonist resulted in an increase in proliferation, which was blocked with the H4R-specific antagonist JNJ7777120. Abdominal epidermis of H4R-deficient mice was significantly thinner, and the in vitro proliferation of keratinocytes derived from H4R-deficient mice was lower compared with that seen in control mice. Interestingly, we only detected H4R expression on murine keratinocytes after stimulation with LPS and peptidoglycan. Conclusion H4R is highly expressed on keratinocytes from patients with atopic dermatitis, and its stimulation induces keratinocyte proliferation. This might represent a mechanism that contributes to the epidermal hyperplasia observed in patients with atopic dermatitis.

Histamine down-regulates IL-27 production in monocytes, and stimulation of keratinocytes with supernatants from histamine-treated monocytes down-regulates CXCL10 secretion. Histamine is a potent mediator in allergic inflammation with immunomodulatory properties. Since histamine was described to inhibit IL-12 production in human APCs, we hypothesized that also the expression of IL-27, a newly described member of the IL-12 family, which is present in inflammatory skin lesions, is modulated by histamine. Stimulation of human monocytes with histamine resulted in significant reduction of TLR ligand-induced IL-27 production in human monocytes. IL-27 subunits, p28 and EBI3, were down-regulated at the mRNA and protein level, whereas other cytokines, such as IL-6, IL-10, and TNF-α, were not influenced. Studies with histamine receptor-specific agonists and antagonists showed that the down-regulation of IL-27 was mediated via H2R and H4R but not H1R and H3R. Human KCs treated with supernatants of histamine-prestimulated monocytes induced significantly less CXCL10 than supernatants containing high levels of IL-27. DCs from H4R−/− mice responded to TLR simulation with higher IL-27 production as compared with WT mice. The down-regulation of IL-27 by histamine might be a new mechanism in the pathogenesis of inflammatory skin diseases, in particular, if increased concentrations of histamine are present at sites of inflammation, such as in chronic eczema and psoriasis.

Plasmacytoid dendritic cells (pDC) are present in inflammatory skin lesions, in particular, in psoriasis. In such lesions, the inflammatory mediator histamine is also detected in high amounts. We therefore investigated a possible interaction of pDC with histamine, especially via the most recently described histamine H(4) receptor (H(4)R). We detected the expression of the H(4)R on pDC in the blood and in lesional psoriasis skin. Interestingly, compared with healthy controls and patients with atopic dermatitis, pDC from the blood of psoriasis patients expressed the highest levels of the H(4)R, which was even more upregulated on stimulation with IFN-γ and CpG. After activation of the H(2)R and H(4)R on pDC, we observed downregulation of CpG-induced production of tumor necrosis factor α, IFN-α, and CXCL8, but not of the chemokine CXCL10. Histamine-induced downregulation of cytokine production was more pronounced in pDC derived from psoriasis patients. Furthermore, we observed F-actin polymerization and active migration of pDC in response to H(4)R agonist stimulation. Taken together, our results indicate that the H(4)R is highly expressed on pDC in psoriasis and influences cytokine production and migration of pDC. Therefore, the H(4)R alone or in combination with the H(2)R might be a promising therapeutic target in psoriasis.

Histamine has long been recognised as a classical inducer of pruritus. However, the specific mechanism of histamine-induced itch has still not been fully understood. The H1 and H4 receptor appear to be key components in the induction of itch. The specific role of the H3 receptor in histamine-induced itch remains unclear. The aim of our study was to investigate the role of the four known histamine receptors (H1-4) in acute itch in mice. Intradermal injection of the selective H3R inverse agonist pitolisant induced strong itch in mice. Pitolisant (50 nmol/injection)-induced pruritus could be completely blocked by a combined treatment with the H1R antagonist cetirizine (15 mg/kg) and the H4R antagonist JNJ 7777120 (15 mg/kg), whereas the H2R antagonist ranitidine (15 mg/kg) failed to inhibit the scratch response. Next, expression and function of histamine receptors on sensory neurons isolated from dorsal root ganglia of mice were investigated. As the itch sensation results from the excitation of sensory nerves in the skin, we further focused on skin specific sensory neurons. Therefore, neurons were retrograde labelled from the skin by means of a fluorescent tracer. Expression of H1R, H3R and H4R on skin innervating sensory neurons was detected. By single-cell calcium imaging, it was demonstrated that histamine induces a calcium increase in a subset of (skin-specific) sensory neurons via activation of the H1R and H4R as well as inhibition of the H3R. It is assumed that the decreased threshold in response to H3R antagonism activates H1R and H4R on sensory neurons, which in turn results in the excitation of histamine-sensitive afferents and therefore elicits the sensation of itch.

Background Histamine influences T-cell reactions via histamine receptors 1 and 2. The histamine receptor 4 (H 4 R) is the most recently identified histamine receptor and is also expressed on human CD4 + T cells; however, its regulation and function are unclear. Objective To investigate expression, regulation, and function of the H 4 R on human CD4 + T cells. Methods Histamine receptor 4 expression was studied by real-time quantitative RT-PCR and by flow cytometry. Effects of H 4 R stimulation on induction of the signal transduction molecules activator protein 1 (AP-1) and nuclear factor-κB (NF-κB) were determined by electrophoretic mobility shift assay and on cytokine production by RT-PCR and ELISA. Results Histamine receptor 4 mRNA and protein were expressed by CD4 + T cells and upregulated by IL-4. Its expression was higher on T H 2 cells than T H 1 cells and naive T-cells. H 4 R agonists (clobenpropit and 4-methylhistamine) induced AP-1 in T H 2 cells but not in T H 1 cells. This effect was blocked by the H 4 R antagonist JNJ7777120. H 4 R agonists upregulated IL-31 mRNA in PBMCs and T H 2 cells, a cytokine that has been associated with T H 2 cells and the induction of pruritus. IL-31 mRNA induction by H 4 R stimulation was pronounced in PBMCs from patients with atopic dermatitis. Expression of IL-4, IL-5, and IL-13 was not altered by the H 4 R. Conclusion Human CD4 + T cells express a functional H 4 R. The receptor is upregulated under T H 2 conditions, and its stimulation leads to induction of AP-1 and IL-31.

Chronic inflammatory skin diseases such as atopic dermatitis (AD) are clinically characterized by erythematous and pruritic skin lesions, immunologically mediated by an inflammatory infiltrate consisting of T-cells, antigen presenting cells (APC) and eosinophilic granulocytes. Histamine levels are increased in lesions of inflammatory skin diseases. It is likely that histamine also plays a pathogenetic role since various relevant cell types such as T-cells and APC express functional histamine receptors. However, therapeutic blockade of the histamine H1 and H2 receptor is inefficient at least in the treatment of atopic dermatitis. We summarize here current data on the role of the recently described histamine H4 receptor (H4R) in chronic inflammatory skin diseases. The H4R is functionally expressed on relevant cell types such as T-cells, APC and keratinocytes. In murine models of contact hypersensitivity and pruritus, H4R blockade had significant in vivo effects. Taken together, several lines of evidence suggest a role of the H4R in chronic inflammatory skin disease and the H4R might be a therapeutic target for diseases such as AD.