RESUMO
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.
Assuntos
Autoantígenos/metabolismo , Derme/metabolismo , Epiderme/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Adulto , Idoso , Células Cultivadas , Feminino , Fibroblastos/metabolismo , Humanos , Queratinócitos/metabolismo , Masculino , Melanócitos/metabolismo , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Pele/metabolismo , Adulto JovemRESUMO
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.
Assuntos
Acantólise/genética , Desmossomos/fisiologia , Epiderme/patologia , Queratinócitos/fisiologia , Proteínas de Membrana/metabolismo , Mucosa Bucal/patologia , Adesão Celular , Diferenciação Celular , Células Cultivadas , Desmogleínas/metabolismo , Epiderme/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/genética , Mucosa Bucal/metabolismo , Técnicas de Cultura de Órgãos , RNA Interferente Pequeno/genéticaRESUMO
Skin resident T cells provide immediate immunologic responses at their specific location and play a role in the pathogenesis of skin diseases such as psoriasis. Recently, IL-9-producing T cells were described as a major T-cell subtype present in the skin, but knowledge on the biology and in situ regulation of this T-cell subtype is scarce. Here, we investigated the cytokine influence on skin T cells with focus on IL-9-producing T cells because a better understanding of their biology may identify novel therapeutic approaches. Healthy human skin biopsies were cultured either in the presence of IL-2, IL-4, and TGF-ß [T helper (Th)9-promoting condition (Th9-PC)] or IL-2 and IL-15 [standard condition (SC)]. Paired analysis of enzymatically isolated skin T cells and emigrated T cells after 4 wk of skin culture showed significant alterations of T-cell phenotypes, cytokine production, and IL-9-producing T-cell frequency. RNA sequencing analysis revealed differentially regulated pathways and identified CXCL8 and CXCL13 as top up-regulated genes in Th9-PC compared with SC. Functionally supernatant of stimulated skin-derived T cells, CXCL8 and CXCL13 increased neutrophil survival. We report that the cytokine environment alters skin-derived T-cell phenotype and functional properties.-Kienzl, P., Polacek, R., Reithofer, M., Reitermaier, R., Hagenbach, P., Tajpara, P., Vierhapper, M., Gschwandtner, M., Mildner, M. Jahn-Schmid, B., Elbe-Bürger, A. The cytokine environment influence on human skin-derived T cells.
Assuntos
Citocinas/imunologia , Regulação da Expressão Gênica/imunologia , Psoríase/imunologia , Pele/imunologia , Linfócitos T/imunologia , Técnicas de Cultura de Células , Células Cultivadas , Feminino , Humanos , Masculino , Psoríase/patologia , Pele/patologia , Linfócitos T/patologiaRESUMO
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.
Assuntos
Alérgenos/imunologia , Apresentação de Antígeno , Betula/imunologia , Hipersensibilidade/imunologia , Imunoglobulina E/imunologia , Neutrófilos/imunologia , Pólen/imunologia , Rinite Alérgica Sazonal/imunologia , Citocinas/imunologia , Humanos , Linfócitos T/imunologiaRESUMO
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.
Assuntos
Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Vesículas Extracelulares , Raios gama , Leucócitos Mononucleares/efeitos da radiação , Neovascularização Fisiológica/efeitos dos fármacos , Proteoma , Células A549 , Animais , Células Cultivadas , Fracionamento Químico , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/efeitos da radiação , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteoma/química , Proteoma/metabolismo , Proteoma/farmacologia , Proteoma/efeitos da radiação , Via Secretória/efeitos da radiação , Cicatrização/efeitos dos fármacosRESUMO
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.
Assuntos
Folículo Piloso/citologia , Queratinócitos/citologia , Antígenos Transformantes de Poliomavirus/biossíntese , Antígenos Transformantes de Poliomavirus/genética , Linhagem Celular , Folículo Piloso/metabolismo , Humanos , Queratinócitos/metabolismo , Telomerase/biossíntese , Telomerase/genéticaRESUMO
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.
Assuntos
Células Dendríticas/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular/imunologia , Linhagem Celular , Separação Celular , Células Dendríticas/citologia , Células Dendríticas/imunologia , Derme/citologia , Derme/imunologia , Derme/metabolismo , Retículo Endoplasmático/imunologia , Células Epidérmicas/imunologia , Células Epidérmicas/metabolismo , Epiderme/imunologia , Epiderme/metabolismo , Sangue Fetal/citologia , Citometria de Fluxo , Voluntários Saudáveis , Células-Tronco Hematopoéticas , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/imunologia , Cultura Primária de Células , Isomerases de Dissulfetos de Proteínas/metabolismo , Isoformas de Proteínas/imunologia , RNA Mensageiro/metabolismoRESUMO
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)-1ß. 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.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Caspase 1/metabolismo , Epiderme/patologia , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Líquen Plano/patologia , Biópsia , Proteínas Adaptadoras de Sinalização CARD/genética , Diferenciação Celular/fisiologia , Regulação para Baixo , Células Epidérmicas , Epiderme/metabolismo , Imunofluorescência , Técnicas de Silenciamento de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Queratinócitos/fisiologia , Psoríase/patologia , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Técnicas de Cultura de TecidosRESUMO
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.
Assuntos
Ácidos Docosa-Hexaenoicos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Inflamação/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Western Blotting , Cromatografia Líquida de Alta Pressão , Fibroblastos/metabolismo , Humanos , Técnicas Imunoenzimáticas , Peroxidação de Lipídeos/efeitos dos fármacos , Peroxidação de Lipídeos/fisiologia , Peroxidação de Lipídeos/efeitos da radiação , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/efeitos da radiação , Espectrometria de Massas em Tandem , Raios Ultravioleta/efeitos adversosRESUMO
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.
Assuntos
Expressão Gênica , Glucuronidase/genética , Gliceraldeído-3-Fosfato Desidrogenases/genética , Queratinócitos , Microglobulina beta-2/genética , Actinas/genética , Biomarcadores , Biomarcadores Tumorais/genética , Diferenciação Celular , Biologia Computacional/métodos , Bases de Dados Genéticas , Complexo II de Transporte de Elétrons/genética , Humanos , Integrina alfa6/genética , Queratina-5/genética , Lectinas/genética , Proteínas de Neoplasias/genética , Fosfoglicerato Quinase/genética , Psoríase/genética , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Ribossômicas/genética , Proteína A7 Ligante de Cálcio S100 , Proteínas S100/genética , Software , Receptores Toll-Like/genética , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
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)-1ß 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.
Assuntos
Defensinas/fisiologia , Bactérias Gram-Negativas , Pleura/imunologia , Líquidos Corporais/química , Defensinas/análise , Drenagem , Humanos , Período Pós-OperatórioRESUMO
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 ß-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.
Assuntos
Peptídeos Catiônicos Antimicrobianos/biossíntese , Feto/imunologia , Histonas/metabolismo , Queratinócitos/imunologia , Células Cultivadas , Humanos , Histona Desmetilases com o Domínio Jumonji/fisiologia , Metilação , Proteínas S100/biossíntese , Proteínas S100/genética , Receptores Toll-Like/fisiologia , beta-Defensinas/biossíntese , beta-Defensinas/genéticaRESUMO
BACKGROUND: Histamine is an important mediator of allergic diseases. It modulates the cytokine expression of various subtypes of antigen-presenting cells by four known receptors, H1R-H4R. The effects of histamine on myeloid dendritic cells (mDC) are unclear. METHODS: Monocytes and mDC were isolated from human PBMC. Histamine receptor expression was evaluated by real-time PCR. Cells were stimulated with histamine and histamine receptor ligands, and restimulated with polyinosinic-polycytidylic acid (poly I:C), and supernatants were analyzed by protein array and ELISA. RESULTS: Monocytes and mDC express H1R and H2R without significant differences between the two cell types, whereas H4R mRNA was significantly higher in mDC compared with monocytes and H3R mRNA was not detected in any cell type. Prestimulation with histamine caused a significant decrease in poly I:C-induced expression of interferon-γ-induced protein (IP-10) in mDC and monocytes. Stimulation with specific H1R, H2R and H4R agonists and antagonists showed that the observed effect was mediated via H2R and H4R in monocytes and mDC. CONCLUSION: Monocytes and mDC have similar histamine receptor repertoires with regard to H1R, H2R and H3R, but H4R expression is higher on mDC. Histamine stimulation shows similar functional effects on both cell types, i.e., downregulation of TLR3-induced IP-10 production. This might be a new mechanism how histamine fosters a Th2 milieu.
Assuntos
Quimiocina CXCL10/antagonistas & inibidores , Células Dendríticas/efeitos dos fármacos , Histamina/farmacologia , Monócitos/efeitos dos fármacos , Quimiocina CXCL10/genética , Quimiocina CXCL10/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interferon gama/biossíntese , Interferon gama/imunologia , Monócitos/citologia , Monócitos/imunologia , Especificidade de Órgãos , Poli I-C/farmacologia , Cultura Primária de Células , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/imunologia , Receptores Histamínicos/genética , Receptores Histamínicos/imunologia , Receptores Histamínicos H1/genética , Receptores Histamínicos H1/metabolismo , Receptores Histamínicos H2/genética , Receptores Histamínicos H2/metabolismo , Receptores Histamínicos H3/deficiência , Receptores Histamínicos H3/genética , Receptores Histamínicos H4 , Equilíbrio Th1-Th2/efeitos dos fármacosAssuntos
Quimiocina CCL27/genética , Quimiocina CCL27/imunologia , Epiderme/imunologia , Queratinócitos/citologia , Queratinócitos/imunologia , Adulto , Diferenciação Celular/imunologia , Quimiocina CCL27/metabolismo , Epiderme/embriologia , Epiderme/crescimento & desenvolvimento , Humanos , Queratinócitos/metabolismoRESUMO
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.
Assuntos
Dermatite Atópica/imunologia , Queratinócitos/imunologia , Receptores Acoplados a Proteínas G/biossíntese , Receptores Histamínicos/biossíntese , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Histamina/imunologia , Antagonistas dos Receptores Histamínicos/farmacologia , Humanos , Indóis/farmacologia , Queratinócitos/efeitos dos fármacos , Lipopolissacarídeos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Peptidoglicano/imunologia , Piperazinas/farmacologia , Receptores Acoplados a Proteínas G/genética , Receptores Histamínicos/genética , Receptores Histamínicos H4RESUMO
Non-healing skin ulcers are often resistant to most common therapies. Treatment with growth factors has been demonstrated to improve closure of chronic wounds. Here we investigate whether lyophilized culture supernatant of freshly isolated peripheral blood mononuclear cells (PBMC) is able to enhance wound healing. PBMC from healthy human individuals were prepared and cultured for 24 hours. Supernatants were collected, dialyzed and lyophilized (SEC(PBMC)). Six mm punch biopsy wounds were set on the backs of C57BL/6J-mice and SEC(PBMC) containing emulsion or controls were applied daily for three days. Morphology and neo-angiogenesis were analyzed by H&E-staining and CD31 immuno-staining, respectively. In vitro effects on diverse skin cells were investigated by migration assays, cell cycle analysis, and tube formation assay. Signaling pathways were analyzed by Western blot analysis. Application of SEC(PBMC) on 6 mm punch biopsy wounds significantly enhanced wound closure. H&E staining of the wounds after 6 days revealed that wound healing was more advanced after application of SEC(PBMC) containing emulsion. Furthermore, there was a massive increase in CD31 positive cells, indicating enhanced neo-angiogenesis. In primary human fibroblasts (FB) and keratinocytes (KC) migration but not proliferation was induced. In endothelial cells (EC) SEC(PBMC) induced proliferation and tube-formation in a matrigel-assay. In addition, SEC(PBMC) treatment of skin cells led to the induction of multiple signaling pathways involved in cell migration, proliferation and survival. In summary, we could show that emulsions containing the secretome of PBMC derived from healthy individuals accelerates wound healing in a mouse model and induce wound healing associated mechanisms in human primary skin cells. The formulation and use of such emulsions might therefore represent a possible novel option for the treatment of non-healing skin ulcers.
Assuntos
Leucócitos Mononucleares/citologia , Cicatrização/fisiologia , Animais , Western Blotting , Movimento Celular/fisiologia , Células Cultivadas , Fibroblastos/citologia , Humanos , Imuno-Histoquímica , Queratinócitos/citologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
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.
Assuntos
Dermatite Atópica/tratamento farmacológico , Dermatite Atópica/imunologia , Epiderme/efeitos dos fármacos , Epiderme/imunologia , Antagonistas dos Receptores Histamínicos/farmacologia , Administração Tópica , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Cimetidina/farmacologia , Dermatite de Contato/tratamento farmacológico , Dermatite de Contato/imunologia , Difenidramina/farmacologia , Modelos Animais de Doenças , Epiderme/metabolismo , Feminino , Antagonistas dos Receptores Histamínicos H1/farmacologia , Antagonistas dos Receptores H2 da Histamina/farmacologia , Homeostase/efeitos dos fármacos , Homeostase/imunologia , Irritantes/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/imunologia , Camundongos , Camundongos Pelados , Permeabilidade/efeitos dos fármacosRESUMO
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 H(2)R and H(4)R but not H(1)R and H(3)R. Human KCs treated with supernatants of histamine-prestimulated monocytes induced significantly less CXCL10 than supernatants containing high levels of IL-27. DCs from H(4)R(-/-) 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.
Assuntos
Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/metabolismo , Agonistas dos Receptores Histamínicos/farmacologia , Histamina/farmacologia , Interleucinas/metabolismo , Receptores Histamínicos/fisiologia , Animais , Western Blotting , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Regulação para Baixo , Ensaio de Imunoadsorção Enzimática , Humanos , Interferon gama/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-12/genética , Interleucina-12/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucinas/genética , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
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-1ß 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.
Assuntos
Dermatite Atópica/imunologia , Receptores Acoplados a Proteínas G/fisiologia , Receptores Histamínicos/fisiologia , Células Th17/metabolismo , Diferenciação Celular , Combinação de Medicamentos , Humanos , Interleucina-17/biossíntese , Interleucina-1beta/farmacologia , Interleucina-23/farmacologia , Metilistaminas/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Receptores Histamínicos/metabolismo , Receptores Histamínicos H4 , Células Th17/patologia , Fator de Transcrição AP-1/farmacologiaRESUMO
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.