Search

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.

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.

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.

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.

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.

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.

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.

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.