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PURPOSE: We previously showed that autologous dendritic cells (DCs) loaded with an allogeneic heat shock (HS)-conditioned melanoma cell-derived lysate, called TRIMEL, induce T-cell-mediated immune responses in stage IV melanoma patients. Importantly, a positive delayed-type hypersensitivity (DTH) reaction against TRIMEL after vaccination, correlated with patients prolonged survival. Furthermore, we observed that DTH reaction was associated with a differential response pattern reflected in the presence of distinct cell subpopulations in peripheral blood. Detected variations in patient responses encouraged molecular studies aimed to identify gene expression profiles induced after vaccination in treated patients, allowing the identification of new molecular predictive markers. METHODS: Gene expression patterns were analyzed by microarrays during vaccination, and some of them confirmed by quantitative real-time reverse transcriptase PCR (qRT-PCR) in the total leukocyte population of a representative group of responder and non-responder patients. New candidates for biomarkers with predictive value were identified using bioinformatics, molecular analysis, and flow cytometry. RESULTS: Seventeen genes overexpressed in responder patients after vaccination respect to non-responders were identified after a mathematical analysis, from which ten were linked to immune responses and five related to cell cycle control and signal transduction. In immunological responder patients, increased protein levels of the chemokine receptor CXCR4 and the Fc-receptor CD32 were observed on cell membranes of CD8+ T and B cells and the monocyte population, respectively, confirming gene expression results. CONCLUSIONS: Our study contributes to finding new molecular markers associated with clinical outcome and better understanding of clinically relevant immunological responses induced by anti-tumor DC-vaccines.
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Psoriasis is an inflammatory skin disease with or without joint involvement. In this disease, the thickened epidermis and impaired barrier are associated with altered calcium gradients. Calcium and vitamin D are known to play important roles in keratinocyte differentiation and bone metabolism. Intracellular calcium is regulated by calcium-sensing receptor (CASR), calcium release-activated calcium modulator (ORAI) and stromal interaction molecule (STIM). Other proteins modulated by vitamin D play important roles in calcium regulation e.g., calbindin 1 (CALB1) and transient receptor potential cation channel 6 (TRPV6). In this study, we aimed to investigate the expression of calcium-regulating proteins in the plaques of patients with psoriasis vulgaris with or without joint inflammation. We confirmed low calcium levels, keratinocyte hyperproliferation and an altered epidermal barrier. The CASR, ORAI1, ORAI3, STIM1, CALB1 and TRPV6 mRNA, as well as the sterol 27-hydroxylase (CYP27A1), 25-hydroxyvitamin D3 1-α-hydroxylase (CYP27B1) and 1,25-dihydroxyvitamin D3 24-hydroxylase (CYP24A1) protein levels were low in the plaques of patients with psoriasis. We demonstrated S100 calcium-binding protein A7 (S100A7) overexpression in the plaques of patients with psoriasis vulgaris with joint inflammation, compared with those without joint involvement. We suggest an altered capacity to regulate the intracellular Ca2+ concentration ([Ca2+]i), characterized by a reduced expression of CASR, ORAI1, ORAI3, STIM1, CALB1 and TRPV6 associated with diminished levels of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], which may be associated with an altered balance between keratinocyte proliferation and differentiation in the psoriatic epidermis. Additionally, differences in S100A7 expression depend on the presence of joint involvement.
Assuntos
Cálcio/metabolismo , Articulações/patologia , Psoríase/genética , Proteínas S100/metabolismo , Vitamina D/farmacologia , Biomarcadores/metabolismo , Diferenciação Celular , Proliferação de Células , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica , Humanos , Queratinócitos/patologia , Pessoa de Meia-Idade , Psoríase/enzimologia , Psoríase/patologia , Receptores de Calcitriol/metabolismo , Proteína A7 Ligante de Cálcio S100RESUMO
BACKGROUND: Psoriasis, a chronic skin disease with or without joint inflammation, has increased circulating proinflammatory cytokine levels. Vitamin D is involved in calcium homeostasis, bone formation, osteoclastogenesis and osteoclast activity, as well as regulation of immune response. We aimed to study osteoclast differentiation and cytokine secretion of peripheral blood mononuclear cells (PBMCs) from patients with psoriasis vulgaris and psoriatic arthritis, in response to 1,25(OH)2D3. METHODS: Serum levels of bone turnover markers were measured by ELISA in patients with psoriasis vulgaris and psoriatic arthritis, and healthy controls. PBMCs were isolated and cultured with or without RANKL/M-CSF and 1,25(OH)2D3. Osteoclast differentiation and cytokine secretion were assessed. RESULTS: Psoriatic arthritis patients had lower osteocalcin, as well as higher C-telopeptide of type I collagen and cathepsin K serum levels compared with psoriasis vulgaris patients and controls. RANKL/M-CSF-stimulated PBMCs from psoriatic arthritis patients produced higher proinflammatory cytokine levels and had a differential secretion profile in response to 1,25(OH)2D3, compared with psoriasis vulgaris and control PBMCs. CONCLUSIONS: Our data confirmed altered bone turnover in psoriatic arthritis patients, and demonstrated increased osteoclastogenic potential and proinflammatory cytokine secretion capacity of these PBMCs compared with psoriasis vulgaris and controls. 1,25(OH)2D3 abrogated these effects.
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Artrite Psoriásica/sangue , Calcitriol/farmacologia , Monócitos/efeitos dos fármacos , Psoríase/sangue , Calcitonina/sangue , Cálcio/sangue , Citocinas/metabolismo , Humanos , Osteoclastos/patologiaRESUMO
OBJECTIVES: Behçet's disease (BD) is an immune-mediated small vessel systemic vasculitis. Human ß-defensins are antimicrobial peptides associated with many inflammatory diseases and are encoded by the ß-defensin family of multiple-copy genes. However, their role in BD necessitates further investigation. The aim of the present study was to investigate the possible association of BD in its various clinical forms with defensin ß-4 (DEFB4) genomic copy numbers. METHODS: This case-control study was conducted from January to September 2011 and included 50 control subjects and 27 unrelated Iraqi BD patients registered at Baghdad Teaching Hospital, Bagdad, Iraq. Copy numbers of the DEFB4 gene were determined using the comparative cycle threshold method by duplex real-time polymerase chain reaction technology at the Department of Dermatology of Jena University Hospital, Jena, Germany. RESULTS: DEFB4 genomic copy numbers were significantly higher in the BD group compared to the control group (P = 0.010). However, no statistically significant association was found between copy numbers and clinical variables within the BD group. CONCLUSION: The DEFB4 copy number polymorphism may be associated with BD; however, it is not associated with different clinical manifestations of the disease.
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Dermatophytes initiate dermatophytosis, but susceptibility to infection is dictated by host genetic factors, although the role of some of these-such as human beta-defensin 2 (hBD-2) genomic (DEFB4) copy number (CN) variation and its induction by IL-22-remains unclear. This was investigated in this cross-sectional study in 442 unrelated Caucasian subjects, including 195 healthy controls and 247 dermatophytosis patients who were divided into five subgroups according to clinical presentation. DNA samples were evaluated for DEFB4 CN variation by relative quantification using the comparative CT method, and serum hBD-2 and IL-22 levels were determined by ELISA. DEFB4 CN in patients was significantly lower and, except in the tinea cruris subgroup, serum hBD-2 levels were higher than in controls. The positive correlation between hBD-2 levels and DEFB4 CN observed in controls was not detected in patients, who also had higher serum IL-22 levels that were positively correlated with hBD-2 levels. Moreover, unlike in control subjects, the serum IL-22 level was negatively correlated with DEFB4 CN in patients. Taken together, these findings suggest an association between decreased DEFB4 CN, elevated serum hBD-2 and IL-22 levels, and dermatophytosis, underscoring a gene/cytokine interaction in the occurrence of this infection.
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Dosagem de Genes/genética , Interleucinas/sangue , Tinha/sangue , Tinha/genética , beta-Defensinas/sangue , beta-Defensinas/genética , Adolescente , Adulto , Idoso , Biomarcadores/sangue , Estudos de Casos e Controles , Estudos Transversais , Feminino , Predisposição Genética para Doença/genética , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Tinha/microbiologia , Trichophyton , Adulto Jovem , Interleucina 22RESUMO
BACKGROUND & AIMS: Reduced epidermal ceramide content may lead to an impaired skin barrier in atopic dermatitis. Plasma concentration of the ceramide precursor sphingomyelin increases after milk-fat consumption due to affected lipoprotein metabolism, although sphingomyelin, a main component of milk phospholipids, might also directly influence plasma sphingomyelin levels. The aim was to determine whether supplementation of a dairy drink with milk phospholipids improves skin parameters and influences plasma lipid profile. METHODS: In a double-blind cross-over study, 39 patients were randomized into 2 groups and daily received phospholipid milk (3 g phospholipids â 0.75 g sphingomyelin) or normal whole milk as placebo control for 6 weeks. SCORAD indices, serum immune and plasma lipid parameters were determined. RESULTS: SCORAD indices did not differ between groups following control and phospholipid milk supplementation (control milk: 10.9 ± 5.9 vs. phospholipid milk: 11.7 ± 6.9, P = 0.416), but were significantly decreased compared to baseline (baseline: 15.6 ± 8.8, P < 0.05). Plasma sphingomyelin proportions were also similar after the treatments (control milk: 27.5 ± 2.3 vs. phospholipid milk: 27.4 ± 2.6% of total phospholipids, P = 0.894), but were significantly increased compared to baseline (20.7 ± 2.4% of total phospholipids, P < 0.05). CONCLUSIONS: Supplementation of a dairy drink with milk phospholipids has no beneficial effect on skin parameters compared to consumption of whole milk in patients with atopic dermatitis. To elucidate an impact of the plasma sphingomyelin proportion on skin conditions, further studies are necessary. Clinical trial ID: Registered under ClinicalTrials.gov. Identifier no. NCT01326520.
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Laticínios , Dermatite Atópica/dietoterapia , Alimentos Fortificados , Fosfolipídeos/administração & dosagem , Adulto , Proteína C-Reativa/metabolismo , Quimiocina CCL22/sangue , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Estudos Cross-Over , Método Duplo-Cego , Selectina E/sangue , Feminino , Humanos , Imunoglobulina E/sangue , Interleucina-16/sangue , Masculino , Esfingomielinas/sangue , Inquéritos e Questionários , Resultado do Tratamento , Triglicerídeos/sangue , Adulto JovemRESUMO
We recently developed a native multidimensional chromatographic method for serum and plasma fractionation for proteomic biomarker search. This method has several advantages:parallelization and automation, high reproducibility and proteome coverage, flexible dynamic range with respect to molecular weight and sample amount, optional enzymatic and immunological analytics additional to mass spectrometry, retaining metabolites, and information on complex formation, modification, and fragmentation of constituents. Nevertheless, native conditions have the probable risk of proteome alteration and biomarker loss by intrinsic proteinases. Hence, we tried to quantify here intrinsic proteolytic activity in native samples and fractions from serum, plasma and cerebrospinal fluid, as well as the effectiveness of intrinsic anti-proteinases during sample handling and preparation under our fractionation conditions. Therefore, we used several quantitative measures: (1) total proportion of intrinsic protein and peptide fractions, (2) azocasein hydrolysis and (3) mass spectrometric protein coverage and peptide numbers. To 1: In all non-fractionated specimens, neither decrease of protein concentration or molecular weight nor increase of peptide concentration was found after variable clotting or pre-incubation time. To 2: No azocasein hydrolysis was seen in these samples when prepared within a few hours at room temperature. Trypsin, when added in concentrations not higher than 0.85 µg/mL (0.04 µM), even was completely inhibited. Moreover, in native 1-D fractions no proteinase activity could be observed. To 3: Mass spectrometry confirmed that neither protein coverage nor peptide numbers differ significantly in 1-D or 2-D fractions after variable incubation time. These results suggest that intrinsic, native proteinase inhibitors potentially protect the proteomes considered, enabling "top-down" proteomic approaches under native conditions with serum, plasma and cerebrospinal fluid.
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Caseínas/sangue , Caseínas/líquido cefalorraquidiano , Cromatografia em Gel/métodos , Cromatografia por Troca Iônica/métodos , Ensaios Enzimáticos Clínicos/métodos , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/líquido cefalorraquidiano , Adulto , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Biomarcadores/química , Caseínas/química , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/química , Proteólise , Psoríase/sangue , Psoríase/líquido cefalorraquidiano , Psoríase/enzimologia , Tripsina/metabolismoRESUMO
Cellular senescence is a permanent cell cycle arrest induced by short telomeres or oncogenic stress in vitro and in vivo. Because no single of the established biomarkers can reliably identify senescent cells, the application of new ones may aid the diagnosis of aged cells. Here we show that annexin A5 accumulates at the nuclear envelope during replicative and drug-induced cellular senescence in primary human fibroblasts. This new cellular aging phenotype that we have termed SA-ANX5 (senescence-associated accumulation at the nuclear envelope of annexin A5) is as efficient and quantitative as the well-established senescence-associated ß-galactosidase activity assay and p21 immunoreactivity. SA-ANX5 is also observed in aged human skin where is exclusively detected in DNA damage foci-positive/Ki-67-negative cells. We also observed that depletion of annexin A5 by siRNA in human fibroblasts accelerates premature senescence through the p38MAP kinase pathway. These observations establish SA-ANX5 as a new biomarker for cellular aging and implicate a functional role for annexin A5 in cellular senescence.
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Anexina A5/metabolismo , Senescência Celular/fisiologia , Fibroblastos/metabolismo , Membrana Nuclear/metabolismo , Pele/metabolismo , Biomarcadores/metabolismo , Dano ao DNA , Fibroblastos/citologia , Células HeLa , Humanos , Antígeno Ki-67/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Pele/citologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The nucleotide adenosine-5'-monophosphate (AMP) can be released by various cell types and has been shown to elicit different cellular responses. In the extracellular space AMP is dephosphorylated to the nucleoside adenosine which can then bind to adenosine receptors. However, it has been shown that AMP can also activate A(1) and A(2a) receptors directly. Here we show that AMP is a potent modulator of mouse and human dendritic cell (DC) function. AMP increased intracellular Ca(2+) concentration in a time and dose dependent manner. Furthermore, AMP stimulated actin-polymerization in human DCs and induced migration of immature human and bone marrow derived mouse DCs, both via direct activation of A(1) receptors. AMP strongly inhibited secretion of TNF-α and IL-12p70, while it enhanced production of IL-10 both via activation of A(2a) receptors. Consequently, DCs matured in the presence of AMP and co-cultivated with naive CD4(+)CD45RA(+) T cells inhibited IFN-γ production whereas secretion of IL-5 and IL-13 was up-regulated. An enhancement of Th2-driven immune response could also be observed when OVA-pulsed murine DCs were pretreated with AMP prior to co-culture with OVA-transgenic naïve OTII T cells. An effect due to the enzymatic degradation of AMP to adenosine could be ruled out, as AMP still elicited migration and changes in cytokine secretion in bone-marrow derived DCs generated from CD73-deficient animals and in human DCs pretreated with the ecto-nucleotidase inhibitor 5'-(alpha,beta-methylene) diphosphate (APCP). Finally, the influence of contaminating adenosine could be excluded, as AMP admixed with adenosine desaminase (ADA) was still able to influence DC function. In summary our data show that AMP when present during maturation is a potent regulator of dendritic cell function and point out the role for AMP in the pathogenesis of inflammatory disorders.
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Actinas/metabolismo , Monofosfato de Adenosina/metabolismo , Cálcio/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Linfócitos T/imunologia , Monofosfato de Adenosina/farmacologia , Análise de Variância , Animais , Movimento Celular/efeitos dos fármacos , Citocinas/metabolismo , Citometria de Fluxo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Polimerização/efeitos dos fármacosRESUMO
Necrobiotic xanthogranuloma (NXG) usually shows a stereotypical histopathologic presentation. However, few unusual cases have been published. We present a patient with NXG showing exceptional histopathologic features. NXG in our patient presents with exclusively dermal granulomatous inflammation mimicking interstitial granuloma annulare. Not only subcutaneous involvement, but also, evident zones of degenerated collagen, foam cells, and cholesterol clefts were missing. Moreover, the case shows overlaps with recently published granulomatous scleromyxedema. Some common clinical and histopathologic features of NXG and scleromyxedema might be based on shared underlying paraproteinemia.
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Xantogranuloma Necrobiótico/patologia , Feminino , Humanos , Imunoglobulinas Intravenosas/uso terapêutico , Pessoa de Meia-Idade , Gamopatia Monoclonal de Significância Indeterminada/complicações , Xantogranuloma Necrobiótico/complicações , Xantogranuloma Necrobiótico/tratamento farmacológicoRESUMO
BACKGROUND: Natural killer (NK) cells have both cytolytic and immunoregulatory functions. We recently described that these cells release the inflammatory cytokines IL-17 and IFN-γ. However, the precise identity of the NK cell subset(s) that secrete these cytokines is not known. METHODOLOGY/PRINCIPAL FINDINGS: To isolate the cells secreting IL-17 and IFN-γ, we took advantage of the findings that Th17/Th1 cells express chemokine receptors. Therefore, CD56(+)NK cells were stained with antibodies against various chemokine receptors and intracellularly with antibodies toward IL-17 and IFN-γ. Consequently, we identified previously unrecognized subset of NK cells generated from normal human peripheral blood after activation with IL-2 but not PMA plus ionomycin. The cells are characterized by the expression of CD56(+) and CCR4(+), produce IL-17 and IFN-γ and are consequently named NK17/NK1 cells. They also express CD161, NKp30, NKp44, NKp46, NKG2D, CD158, CCL22, IL-2Rß and the common γ chain but not CD127 or IL-23R. Further, they possess T-bet and RORγt transcription factors. Antibodies to IL-1ß, IL-6, IL-21, or TGF-ß1 do not inhibit IL-2-induced generation of NK17/NK1 cells, suggesting that IL-2 has the capacity to polarize these cells. Notably, NK17/NK1 cells are abundant in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) without activation, and are generated from the peripheral blood of these patients after activation with IL-2. CONCLUSIONS/SIGNIFICANCE: NK17/NK1 cells identified here have not been previously described in healthy or MS patients.
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Interleucina-17/imunologia , Interleucina-1/imunologia , Células Matadoras Naturais/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Interferon gama/metabolismo , Interleucina-1/metabolismo , Interleucina-17/metabolismo , Células Matadoras Naturais/metabolismo , Esclerose Múltipla/líquido cefalorraquidiano , Esclerose Múltipla/metabolismoRESUMO
PI3Ks control signal transduction triggered by growth factors and G-protein-coupled receptors and regulate an array of biological processes, including cellular proliferation, differentiation, survival and migration. Herein, we investigated the role of PI3Kγ in the pathogenesis of EAE. We show that, in the absence of PI3Kγ expression, clinical signs of EAE were delayed and mitigated. PI3Kγ-deficient myelin oligodendrocyte glycoprotein (MOG)(35-55) -specific CD4(+) T cells appeared later in the secondary lymphoid organs and in the CNS than their WT counterparts. Transfer of WT CD4(+) cells into PI3Kγ(-/-) mice prior to MOG(35-55) immunisation restored EAE severity to WT levels, supporting the relevance of PI3Kγ expression in Th cells for the pathogenesis of EAE; however, PI3Kγ was dispensable for Th1 and Th17 differentiation, thus excluding an altered expression of these pathogenetically relevant cytokines as the cause for ameliorated EAE in PI3Kγ(-/-) mice. These findings demonstrate that PI3Kγ contributes to the development of autoimmune CNS inflammation.
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Classe Ib de Fosfatidilinositol 3-Quinase/deficiência , Encefalomielite Autoimune Experimental/enzimologia , Encefalomielite Autoimune Experimental/prevenção & controle , Transferência Adotiva , Animais , Diferenciação Celular , Classe Ib de Fosfatidilinositol 3-Quinase/genética , Classe Ib de Fosfatidilinositol 3-Quinase/imunologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Glicoproteínas/imunologia , Tecido Linfoide/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Peptídeos/imunologia , Inibidores de Fosfoinositídeo-3 Quinase , Linfócitos T Auxiliares-Indutores/enzimologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/patologia , Fatores de TempoAssuntos
Granuloma/tratamento farmacológico , Imunoglobulinas Intravenosas/administração & dosagem , Transtornos Necrobióticos/tratamento farmacológico , Xantomatose/tratamento farmacológico , Idoso , Biópsia por Agulha , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Seguimentos , Granuloma/patologia , Humanos , Imuno-Histoquímica , Injeções Intravenosas , Pessoa de Meia-Idade , Transtornos Necrobióticos/patologia , Doenças Raras , Índice de Gravidade de Doença , Resultado do Tratamento , Xantomatose/patologiaRESUMO
Members of the Toll/interleukin-1 receptor (TIR) family are of importance for host defense and inflammation. Here we report that the TIR-family member interleukin-33R (IL-33R) cross-activates the receptor tyrosine kinase c-Kit in human and murine mast cells. The IL-33R-induced activation of signal transducer and activator of transcription 3 (STAT3), extracellular signal-regulated kinase 1/2 (Erk1/2), protein kinase B (PKB), and Jun NH(2)-terminal kinase 1 (JNK1) depends on c-Kit and is required to elicit optimal effector functions. Costimulation with the c-Kit ligand stem cell factor (SCF) is necessary for IL-33-induced cytokine production in primary mast cells. The structural basis for this cross-activation is the complex formation between c-Kit, IL-33R, and IL-1R accessory protein (IL-1RAcP). We found that c-Kit and IL-1RAcP interact constitutively and that IL-33R joins this complex upon ligand binding. Our findings support a model in which signals from seemingly disparate receptors are integrated for full cellular responses.
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Proteína Acessória do Receptor de Interleucina-1/metabolismo , Interleucinas/metabolismo , Mastócitos/metabolismo , Proteínas Proto-Oncogênicas c-kit/fisiologia , Receptores de Interleucina/metabolismo , Transdução de Sinais , Animais , Western Blotting , Medula Óssea/metabolismo , Células Cultivadas , Citocinas/metabolismo , Feminino , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Integrases/metabolismo , Proteína 1 Semelhante a Receptor de Interleucina-1 , Interleucina-33 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação , Fator de Células-Tronco/metabolismo , Tirosina/metabolismoRESUMO
Leukocytes are a heterogeneous group of cells that display differences in anatomic localization, cell surface phenotype, and function. The different subtypes include e.g., granulocytes, monocytes, dendritic cells, T cells, B cells and NK cells. These different cell types represent the cellular component of innate and adaptive immunity. Using certain toxins such as pertussis toxin, cholera toxin or clostridium difficile toxin, the regulatory functions of Gα(i), Gαs and small GTPases of the Rho family in leukocytes have been reported. A summary of these reports is discussed in this review.
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Toxinas Bacterianas/farmacologia , Leucócitos/efeitos dos fármacos , Animais , Proteínas de Bactérias/farmacologia , Toxina da Cólera/farmacologia , Enterotoxinas/farmacologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Humanos , Leucócitos/fisiologia , Toxina Pertussis/farmacologia , Receptores Acoplados a Proteínas G/fisiologiaRESUMO
Beside its well described role in the central and peripheral nervous system 5-hydroxytryptamine (5-HT), commonly known as serotonin, is also a potent immuno-modulator. Serotoninergic receptors (5-HTR) are expressed by a broad range of inflammatory cell types, including dendritic cells (DCs). In this study, we aimed to further characterize the immuno-biological properties of serotoninergic receptors on human monocyte-derived DCs. 5-HT was able to induce oriented migration in immature but not in LPS-matured DCs via activation of 5-HTR(1) and 5-HTR(2) receptor subtypes. Accordingly, 5-HT also increased migration of pulmonary DCs to draining lymph nodes in vivo. By binding to 5-HTR(3), 5-HTR(4) and 5-HTR(7) receptors, 5-HT up-regulated production of the pro-inflammatory cytokine IL-6. Additionally, 5-HT influenced chemokine release by human monocyte-derived DCs: production of the potent Th1 chemoattractant IP-10/CXCL10 was inhibited in mature DCs, whereas CCL22/MDC secretion was up-regulated in both immature and mature DCs. Furthermore, DCs matured in the presence of 5-HT switched to a high IL-10 and low IL-12p70 secreting phenotype. Consistently, 5-HT favoured the outcome of a Th2 immune response both in vitro and in vivo. In summary, our study shows that 5-HT is a potent regulator of human dendritic cell function, and that targeting serotoninergic receptors might be a promising approach for the treatment of inflammatory disorders.
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Movimento Celular/fisiologia , Quimiocinas/metabolismo , Citocinas/metabolismo , Células Dendríticas/imunologia , Serotonina/fisiologia , Linfócitos T/imunologia , Animais , Diferenciação Celular , Células Cultivadas , Células Dendríticas/citologia , Humanos , Linfonodos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T/citologiaRESUMO
Lysophosphatidic acid (LPA) is an activator and chemoattractant of NK cells, which are critical members of the immunological tumor surveillance machinery. Here, we analyzed the influence of LPA on the interaction of human NK cells with tumor cells such as the Burkitt lymphoma cell line Raji and the human melanoma cell line A2058. Thereby we found that LPA inhibits the release of perforin and cytotoxic activity of NK cells. Analysis of signal transduction showed that LPA induces common signaling pathways of chemotaxins such as G(i) protein-dependent actin re-organization, activation of the mitogen-activated protein kinase p38 as well as phosphatidylinositol-3-kinase-dependent signal molecules [protein kinase B/Akt and glycogen synthase kinase-3beta (GSK-3beta)]. In contrast to most chemotaxins, LPA is also able to activate G(s)-dependent signaling molecules. This signaling cascade involves the LPA receptor type-2, increase cAMP levels and protein kinase A (PKA) activation, which in turn are responsible for the modulatory effect of LPA on NK cell-mediated cytotoxicity. Moreover, blocking the regulatory subunits of PKA I abrogates the inhibitory effect of LPA, whereas the catalytic subunits are not involved. Based on our data, one can assume that LPA contributes to the tumor escape from the immunological surveillance machinery.
Assuntos
Linfoma de Burkitt/imunologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Células Matadoras Naturais/metabolismo , Lisofosfolipídeos/metabolismo , Melanoma/imunologia , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patologia , Linhagem Celular Tumoral , Classe Ia de Fosfatidilinositol 3-Quinase , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citotoxicidade Imunológica , Regulação para Baixo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/imunologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/imunologia , Humanos , Vigilância Imunológica , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Lisofosfolipídeos/imunologia , Melanoma/metabolismo , Melanoma/patologia , Perforina/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/metabolismo , Receptores de Ácidos Lisofosfatídicos/imunologia , Receptores de Ácidos Lisofosfatídicos/metabolismo , Transdução de Sinais/imunologia , Evasão Tumoral , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
In the treatment of chronic wounds, it is necessary to establish a physiological wound milieu to improve healing. Application of collagen as wound dressing has been described as beneficial as it possesses the ability to reduce elevated levels of proteases, cytokines, and free radicals. Consequently, a wide range of wound dressings based on collagen have been developed. Native collagen is susceptible to alterations because of influences during the production process; to minimize effects on the molecule itself collagen wound dressings are usually aseptically produced. Common sterilization methods (autoclaving, irradiation, and ethylene oxide (EtO) treatment) can induce changes in the protein chemistry and physical properties, potentially affecting the absorption rate, mechanical strength, or performance. In this study, we have evaluated the influence of gamma- and beta-irradiation as well as EtO sterilization on the binding capacity of collagen type I for selected proteases and cytokines associated with nonhealing wounds. Although a pronounced effect on the physical properties of the collagen was found, there was no significant loss in the binding affinity for polymorphonuclear elastase, matrix metalloproteinase-2, and interleukin-1beta, or in the antioxidant capacity.