Assuntos
Fibroblastos/metabolismo , Óxido Nítrico/sangue , Psoríase/metabolismo , Adulto , Idoso , Estudos de Casos e Controles , Linhagem Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Humanos , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Mastócitos/patologia , Pessoa de Meia-Idade , Óxido Nítrico/metabolismo , Psoríase/sangue , Psoríase/patologia , Substância P/farmacologia , Peptídeo Intestinal Vasoativo/farmacologiaRESUMO
The peptide thymosin beta4 (Tbeta4) promotes angiogenesis and wound healing. Mast cells are involved in these processes as well and therefore we investigated the effect of Tbeta4 on mast cells. Exposure to 0.2-2000nM Tbeta4 induced mediator release (up to 23%) in murine peritoneal and human HMC-1 mast cells in a concentration-dependent manner. While the peptide AcSDKP, matching the 4 N-terminal amino acid residues of Tbeta4, mediated low but detectable mediator release, peptides corresponding to the Tbeta4 amino acid sequences 16-38 and 17-23 stimulated mast cells mediator release on a level equal to or higher than that observed with native Tbeta4. These observations and certain characteristics of Tbeta4-mediated mast cell activation suggest that the actin-binding motif LKKTET present in Tbeta4 (amino acid 17-22) might be implicated in this process. Thus, Tbeta4 activates mediator release in mast cells by a process that possibly involves an actin-binding motif and this could be important for understanding the mechanisms of Tbeta4-mediated effects in vivo.
Assuntos
Exocitose/efeitos dos fármacos , Mastócitos/efeitos dos fármacos , Peptídeos/farmacologia , Timosina/farmacologia , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Feminino , Histamina/metabolismo , Humanos , Mastócitos/citologia , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Peptídeos/síntese química , Peptídeos/química , Timosina/química , Fatores de Tempo , Triptases/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
Fibroblasts have been implicated in psoriatic inflammatory processes. The aim of the study was to evaluate soluble interleukin 2 receptor (sIL-2R), interleukin 6 (IL-6), and interleukin 8 (IL-8) plasma levels in psoriatic patients and IL-6 and IL-8 levels in fibroblast culture supernatants. Cytokines levels in plasma and supernatants were measured by ELISA. Plasma sIL-2R, IL-6, and IL-8 levels were higher before the treatment in comparison to healthy controls (P < 0.001) and decreased after treatment. Fibroblasts from healthy controls, psoriatic lesional skin, and noninvolved psoriatic skin, when stimulated with tumor necrosis factor alpha, released considerable amounts of IL-6 and IL-8. No significant difference between healthy controls and psoriatic fibroblasts was observed. Monitoring plasma sIL-2R levels could be employed as a reliable method of psoriasis activity. IL-8 and IL-6 plasma levels seem to reflect psoriasis activity, and treatment response, respectively. Fibroblasts are not a major source of increased IL-6 and IL-8 production in psoriasis.
Assuntos
Fibroblastos/metabolismo , Regulação da Expressão Gênica , Interleucina-6/sangue , Interleucina-8/sangue , Psoríase/sangue , Psoríase/metabolismo , Adulto , Idoso , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Inflamação , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Masculino , Pessoa de Meia-Idade , Receptores de Interleucina-2/sangue , Receptores de Interleucina-2/metabolismoRESUMO
BACKGROUND: Interleukin (IL)-4 plays a prominent role in immune response. Mercuric compounds upregulate IL-4 expression in animal tissues, and this upregulation plays a role in mercuric-mediated immunomodulation. Mercuric ions-mediated IL-4 expression was observed in vitro in T lymphocytes and mast cells. In the present study, we investigated molecular mechanisms responsible for this effect of mercuric ions in mast cells. METHODS: C1.MC/C57.1 mouse mast cells were exposed in vitro to increasing concentrations of Hg(2+) in the absence or presence of the specific c-Jun N-terminal kinase (JNK) inhibitor SP600125. The level of phosphorylated c-Jun in mast cells was determined by Western blotting, JNK activity assessed with in vitro kinase assay and the amount of secreted IL-4 determined by ELISA. RESULTS: We observed that Hg(2+) upregulated c-Jun phosphorylation on Ser 73 at concentrations which overlapped concentrations mediating IL-4 secretion. Phosphorylation of c-Jun in mast cells was associated with an increase in JNK activity. The specific JNK inhibitor SP600125 abolished both mercuric-induced c-Jun phosphorylation and IL-4 secretion in mast cells. CONCLUSIONS: These observations are consistent with the hypothesis that JNK is one of the signaling proteins mediating the effect of Hg(2+) on IL-4 expression in mast cells and is engaged in environmentally mediated immunomodulation.
Assuntos
Interleucina-4/imunologia , Proteínas Quinases JNK Ativadas por Mitógeno/imunologia , Mastócitos/imunologia , Mercúrio/imunologia , Animais , Antracenos/farmacologia , Técnicas de Cultura de Células , Interleucina-4/biossíntese , Íons/imunologia , Proteínas Quinases JNK Ativadas por Mitógeno/efeitos dos fármacos , Mastócitos/efeitos dos fármacos , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Regulação para CimaRESUMO
Predictive testing of immunotoxicity associated with chemical compounds is complicated and cannot be accomplished with a single test. As most of the existing tests for immunotoxicity employ experimental animals, there is an increasing need for alternative tests in vitro. We have developed a new system for in vitro immunotoxicity testing, which employs changes in cytokine expression observed in vitro as an endpoint indicating potential for perturbation of the immune system in vivo. This system named "fluorescent cell chip" (FCC) is based on a number of genetically modified cell lines that regulate the expression of a transgene coding for fluorescent protein enhanced green fluorescent protein (EGFP) in a similar way as they regulate expression of IL-1beta, IL-2, IL-4, IFN-gamma, IL-10, TNF-alpha, and beta-actin. Morphological and functional features of selected cell lines expressing EGFP under the control of cytokine promotors were compared with maternal cell lines and this comparison showed that critical functional features of the maternal cell lines were preserved in EGFP expressing cells. Two chemicals with known immunotoxic activities, cyclosporine A and potassium tetrachloro-platinate(II), mediated compound-specific pattern of inhibition and activation of reporter gene expression. Thus, the "fluorescent cell chip" has demonstrated potential for application as a predictive screening test for immunomodulatory activities of chemicals. The major advantage of this approach is the possibility to apply this test in high throughput screening of high number of compounds for their well defined biological activity.
Assuntos
Citocinas/biossíntese , Proteínas de Fluorescência Verde , Imunotoxinas/toxicidade , Substâncias Luminescentes , Testes de Toxicidade/métodos , Animais , Linhagem Celular Transformada , Linhagem Celular Tumoral , Cloretos/análise , Ciclosporina/análise , Citocinas/imunologia , Proteínas de Fluorescência Verde/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Compostos de Platina/análise , RNA/química , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Psoriasis is regarded as a Th1-cell type disease. Interleukin-4, a Th2 type cytokine, is diminished in psoriatic skin. It has been postulated that switching the cytokine profile from Th1 to Th2 may be of great help in the treatment of psoriasis. Some recent reports demonstrate a favorable role of IL-4 in the treatment of psoriasis. Therefore we decided to evaluate how IL-4 plasma levels fluctuate in the case of favorable treatment outcome observed in patients with clinically active and stable disease. MATERIAL/METHODS: 17 patients with active psoriasis vulgaris, 17 with stable disease, and 22 age- and sex-matched healthy controls were included in the study. IL- 4 plasma levels were evaluated by the ELISA method twice--before treatment implementation and 4 weeks thereafter. RESULTS: We observed statistically significant higher IL-4 plasma levels in the active psoriasis group both before treatment implementation (1st examination) and after 4 weeks of treatment (2nd examination) compared with both the control group (p<0.001) and the stable psoriasis group (1st examination: p<0.001, 2nd examination: p<0.01). There was no statistically significant difference in IL-4 plasma levels between the stable psoriasis and control groups in both examinations (p>0.05). CONCLUSIONS: Our data seem to support the hypothesis of the existence of immunologically distinct psoriasis subtypes.
Assuntos
Interleucina-4/sangue , Psoríase/sangue , Adulto , Antralina/uso terapêutico , Feminino , Ficusina/uso terapêutico , Humanos , Masculino , Metotrexato/uso terapêutico , Pessoa de Meia-Idade , Fototerapia , Psoríase/terapia , Índice de Gravidade de DoençaRESUMO
Thymosin beta 4(T beta 4), a 4.9-kDa polypeptide primarily known as a main G-actin-sequestering peptide, is present in high concentrations in various cells and in the circulation. We have found that T beta 4 upregulates the expression of plasminogen activator inhibitor 1 (PAI-1) in endothelial cells measured both at the level of mRNA and protein synthesis. This effect seems to be cell specific and was not observed when other cells such as human fibroblasts, PC3, and U937 were tested. T beta 4 significantly activated the PAI-1 promoter in EA.hy 926 cells transiently transfected either with plasmid p800LUC containing PAI-1 promoter fragment (-800 to +71) or the PAI-1 promoter linked with green fluorescent protein. T beta 4 mediated up-regulation of PAI-1 involved activation of the mitogen-activated protein kinase cascade. Furthermore, T beta 4 enhanced c-Fos/c-Jun DNA-binding activity to the activator protein 1 (AP-1)-like element (-59 to -52). The specificity of this binding activity was demonstrated by competition electrophoretic mobility shift assay and after transfection of EA.hy 926 cells with the mutated PAI-1 promoter. Taken together, these data indicate that, in response to T beta 4 stimulation, AP-1 activity increases to enhance PAI-1 transcription through its unique AP-1-like element at -59 to -52 in the PAI-1 promoter.
Assuntos
Endotélio Vascular/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Timosina/farmacologia , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Espaço Extracelular/fisiologia , Expressão Gênica/efeitos dos fármacos , Humanos , Mutação Puntual , Regiões Promotoras Genéticas/genética , Fator de Transcrição AP-1/metabolismo , Veias Umbilicais/citologiaRESUMO
Upon contact with allergen, sensitized mast cells release highly active proinflammatory mediators. Allergen-mediated mast cell activation is an important mechanism in the pathogenesis of atopic asthma. Asthmatic patients are especially susceptible to air pollution. Epidemiologic studies found a positive correlation between severity of symptoms among asthmatic patients and the level of particulate matter (PM) in the air. Among the constituents of PM are metals and transition metals, which could mediate some of its adverse effects on human health. We sought to determine the effect of metal and transition metal ions on allergen-mediated mast cell activation. We observed that several metal and transition metal ions activated mast cells and enhanced allergen-mediated mast cell activation. Thus, Al(3+), Cd(2+), and Sr(2+) induced release of granule-associated N-acetyl-ss-d-hexosaminidase, and Al(3+) and Ni(2+) enhanced antigen-mediated release. Metal and transition metal ions also induced significant secretion of interleukin (IL)-4 and increased antigen-mediated IL-4 secretion in mast cells. These effects of metal and transition metal ions on mast cells were observed at concentrations that do not result in direct cytotoxicity and might be relevant for environmental exposure. Thus, metals and transition metals could increase the level of allergen-mediated mast cell activation, which might be one of the mechanisms mediating exacerbation of allergen-driven asthma symptoms by air pollution.