RESUMEN
Patulin is a mold toxin secreted mainly by fungi of the Penicillium species. Exposure generally results from consumption of moldy fruits and fruit products. Since recent studies identified mold exposure as a risk factor for allergic diseases, we examined the effects of patulin on human peripheral blood mononuclear cells (PBMC) prepared from buffy coats of healthy donors. Cells were stimulated with CD3- and CD28-specific antibodies in the presence or absence of patulin. Effects of patulin on PBMCs were evaluated by proliferation, viability assays, and cytokine ELISAs. The presence of 50 ng/mL patulin strongly decreased the amounts of several cytokines in the supernatant of stimulated PBMCs. This decrease in cytokine secretion was not due to cytotoxic effects of patulin. Moreover, the extent of the reduction of cytokine amounts was cytokine specific, affecting some (IL-4, IL-13, IFNgamma, and IL-10), but not others (IL-8, IL-5). We show that all effects could be abolished by adding thiol containing compounds. A depletion of intracellular GSH could be measured after incubation of cells with patulin. Taken together, our data indicate that patulin modulates the functional activation of PBMCs with respect to proliferation and cytokine secretion patterns by depletion of intracellular GSH. The depletion of intracellular glutathione may influence the balance between Th1 and Th2 cells and have implications for allergic diseases.
Asunto(s)
Citocinas/metabolismo , Glutatión/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Micotoxinas/toxicidad , Patulina/toxicidad , Linfocitos T/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Humanos , Leucocitos Mononucleares/metabolismo , Penicillium , Linfocitos T/metabolismo , Células TH1/efectos de los fármacos , Células TH1/metabolismo , Células Th2/efectos de los fármacos , Células Th2/metabolismoRESUMEN
Antibody microarrays have often had limited success in detection of low abundant proteins in complex specimens. Signal amplification systems improve this situation, but still are quite laborious and expensive. However, the issue of sensitivity is more likely a matter of kinetically appropriate microarray design as demonstrated previously. Hence, we re-examined in this study the suitability of simple and inexpensive detection approaches for highly sensitive antibody microarray analysis. N-hydroxysuccinimidyl ester (NHS)- and Universal Linkage System (ULS)-based fluorescein and biotin labels used as tags for subsequent detection with anti-fluorescein and extravidin, respectively, as well as fluorescent dyes were applied for analysis of blood plasma. Parameters modifying strongly the performance of microarray detection such as labeling conditions, incubation time, concentrations of anti-fluorescein and extravidin and extent of protein labeling were analyzed and optimized in this study. Indirect detection strategies whether based on NHS- or ULS-chemistries strongly outperformed direct fluorescent labeling and enabled detection of low abundant cytokines with many dozen-fold signal-to-noise ratios. Finally, particularly sensitive detection chemistry was applied to monitoring cytokine production of stimulated peripheral T cells. Microarray data were in accord with quantitative cytokine levels measured by ELISA and Luminex, demonstrating comparable reliability and femtomolar range sensitivity of the established microarray approach.
Asunto(s)
Anticuerpos , Proteínas Sanguíneas/análisis , Citocinas/análisis , Análisis por Matrices de Proteínas , Coloración y Etiquetado/métodos , Células Cultivadas , Citocinas/metabolismo , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Estructura Molecular , Patulina/farmacologíaRESUMEN
Secretion of various chemokines including Eotaxin-3/CCL26 results in the attraction of eosinophils to sites of allergic inflammation. IL-4/IL-13-induced activation of the Eotaxin-3/CCL26 gene in human dermal fibroblasts was shown to be a STAT6-dependent process mediated by a single STAT6 binding motif located upstream of the transcription initiation site. The suppressors of cytokine signaling 1-3 (SOCS 1-3) are members of a recently discovered family of proteins acting as negative regulators of cytokine signaling. We show here, that transfection of SOCS-1 and SOCS-3 but not SOCS-2 expression vectors inhibited IL-4/IL-13 induced secretion of Eotaxin-3/CCL26. Further, using Eotaxin-3/CCL26 promoter reporter gene constructs, we could show that, upon cotransfection of SOCS-1 and SOCS-3 expression vectors, IL-4 and IL-13 induced luciferase activity was strongly reduced. This effect was not seen when SOCS-2 was cotransfected. Further, EMSA studies with nuclear extracts prepared from IL-4/IL-13 induced HEK293 cells were conducted. The nuclear extracts of cells transfected with SOCS-1 or SOCS-3 did not form complexes with oligonucleotide probes corresponding to the STAT6 binding site in the Eotaxin-3/CCL26 promoter. In contrast, complex formation upon SOCS-2-transfection was comparable to mock-transfected cells. Further, the levels of phosphorylated STAT6 in IL-4 and IL-13 treated cells were markedly reduced when the cells had been transfected with SOCS-1 or SOCS-3, confirming the role of these negative regulators for the IL-4 and IL-13 induced activation of Eotaxin-3/CCL26 gene expression. The insertion of amino acid exchanges into the kinase inhibitory regions of SOCS-1 and SOCS-3 demonstrated a requirement of these domains for a proper inhibitory function.
Asunto(s)
Quimiocinas CC/genética , Interleucina-13/antagonistas & inhibidores , Interleucina-4/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Sustitución de Aminoácidos/genética , Línea Celular , Quimiocina CCL26 , Quimiocinas CC/biosíntesis , Regulación hacia Abajo , Ensayo de Cambio de Movilidad Electroforética , Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Fosforilación , Regiones Promotoras Genéticas/genética , Proteínas Represoras/genética , Elementos de Respuesta/genética , Factor de Transcripción STAT6 , Proteína 1 Supresora de la Señalización de Citocinas , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , TransfecciónRESUMEN
Proteins of the suppressors of cytokine signaling (SOCS) family have important functions as negative regulators of cytokine signaling. We show here that SOCS-1 expression can be induced in the human epithelial lung cell line A549 by IL-4 and IL-13. Analysis of reporter gene constructs under control of the SOCS-1 promoter provides evidence that IL-4- and IL-13-induced up-regulation is dependent on three IFN-gamma-activated sequence motifs of the sequence TTC(N)(4)GAA, which is known for binding STAT6. The three motifs are situated close to each other approximately 600 bp upstream of the transcriptional initiation site. When mutations were inserted into all three IFN-gamma-activated sequence motifs at the same time, IL-4-IL-13-induced luciferase activity was abrogated. With single and double mutants, promoter activity was diminished in comparison with the wild-type promoter. STAT6 is therefore required for IL-4-IL-13-dependent SOCS-1 expression in A549 cells, and the three identified binding motifs cooperate to induce maximal transcription. EMSAs conducted with nuclear extracts of IL-4- and IL-13-stimulated A549 cells showed that STAT6 was able to bind to each of the three binding motifs. Finally, cotransfection of a SOCS-1 expression vector inhibited activation of SOCS-1 promoter luciferase constructs. Thus, SOCS-1 is able to autoregulate its expression via a negative feedback loop.
Asunto(s)
Regiones no Traducidas 5'/fisiología , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Regulación de la Expresión Génica/inmunología , Interleucina-13/fisiología , Interleucina-4/fisiología , Péptidos y Proteínas de Señalización Intracelular , Proteínas Represoras/biosíntesis , Proteínas Represoras/genética , Transactivadores/fisiología , Sitio de Iniciación de la Transcripción , Regiones no Traducidas 5'/metabolismo , Secuencias de Aminoácidos/genética , Secuencias de Aminoácidos/inmunología , Sitios de Unión/genética , Sitios de Unión/inmunología , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/fisiología , Línea Celular Tumoral , Retroalimentación Fisiológica/genética , Retroalimentación Fisiológica/inmunología , Humanos , Interferón gamma/farmacología , Interleucina-13/antagonistas & inhibidores , Interleucina-4/antagonistas & inhibidores , Células Jurkat , Regiones Promotoras Genéticas , Unión Proteica/genética , Unión Proteica/inmunología , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/fisiología , Factor de Transcripción STAT6 , Transducción de Señal/genética , Transducción de Señal/inmunología , Proteína 1 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas , Células Th2/inmunología , Células Th2/metabolismo , Transactivadores/metabolismo , Transfección , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunologíaRESUMEN
Environmental pollutants can influence the expression of immunoregulatory molecules and, in this way, promote allergies. The local synthesis of proinflammatory chemokines is an important aspect in the development of allergic airway inflammation. We have characterized the influence of pyrene, a polycyclic aromatic hydrocarbon (PAH) contained, for example, in diesel exhaust particles (DEP), on transcription and secretion of the chemokines interleukin-8 (IL-8) and eotaxin. Reporter genes under control of the respective promoters were tested in the human cell lines A549 and HeLa, mRNA production was assayed in A549 cells and protein production was measured by ELISA in cell supernatants from primary human fibroblasts. Pyrene content of cell supernatants was measured by analytical HPLC. Promoter activity, mRNA production and protein expression of IL-8 were increased by pyrene. The activating effect in reporter gene studies was abolished by mutating either an NF-kappaB or an AP-1 binding site in the IL-8 promoter. In contrast, pyrene showed no effect on transcription from the eotaxin promoter, despite the important role of this chemokine in asthma. Our data show that pyrene has specific effects on chemokine synthesis, which are not restricted to mediators primarily associated with atopic diseases. Pyrene also affected cells not derived from lung tissue, which suggests a broader immunoregulatory influence for this pollutant.