The research of the Laboratory of Molecular Immunology in 2002 was focused on two major topics. First is the development of a new system for testing immunotoxic effects of xenobiotics in vitro, and the second one is the regulation of cytokine expression in mast cells.
Novel technology for in vitro immunotoxi-city testing
Due to the new chemical safety regulation being now introduced in Europe around 30 000 chemicals will have to be tested to different extend for possible toxicity. This testing will be accomplished not earlier than in 2048, will require 12.8 milions of experimental animals, and is expected to cost 8,64 bilions euro. Therefore there is an increasing demand, for methods suitable for high-throughput screening that increase the speed and reduce the cost per chemical entity for safety evaluation. There are also obvious ethical reasons for implementing in vitro alternatives in safety evaluation in order to reduce, refine, and replace the use of laboratory animals for routine testing. The laboratory coordinated the collaborative effort of six research groups towards development of a new system for testing immunotoxic effects of xenobiotics in-vitro. We have developed a new system for testing immunotoxic effects of chemicals in vitro. Our patent pending technology called "Fluorescent Cell Chip" (FCC) does not involve experimental animals but instead is based on a number of immortalized genetically modified cell lines representing different phenotypes of cells regulating immune response in vivo. FCC consists of a panel of reporter cell lines that regulate the expression of a transgene coding for fluorescent protein in the same way as they regulate expression of cytokines. This test could be employed in a uniform high throughput system for screening tousends of compounds for their possible effect on expression of a number of cytokine genes, which is a well documented and valid endpoint for immunotoxicity. In testing of the prototype of FCC using chemicals with known biological activities 86% of immunotoxic compounds, including skin and pulmonary sensitizers, were positive.
Signaling pathways regulating cytokine expression in mast cells
The second topic of our research is regulation of cytokine expression in mast cells with special emphasis on signal transduction mechanisms engaged in xenobiotic-mediated upregulation of IL-4 and TNF-(alpha) expression. We observed that several metal and transition metal ions induced and enhanced allergen-mediated IL-4 expression. All these effects of metal and transition metal ions on mast cells were observed at concentrations, which might be relevant for the environmental exposure in air pollution.
We investigated the role of JNK in signal transduction mechanisms regulating IL-4 expression in mast cells exposed to mercuric ions. We have gained new evidences for the critical role of JNK in upregulation of IL-4 by antigen and mercuric ions. The employment of a specific JNK inhibitor SP600125 resulted in the complete inhibition of c-Jun phosphorylation and IL-4 secretion. The JNK inhibitor exercised such effect in mast cells activated with antigen or exposed to mercuric chloride or activated concomitantly with both stimuli. These results support the hypothesis that JNK may be a focal point, which allows for a cross talk of two signaling pathways responding to antigen or xenobiotics and regulating cytokine expression in immune cells. Continuing our search for molecular mechanism of mercury-mediated IL-4 expression we investigated effect of mercuric ions on IL-4 promoter activity in mast cells. HgCl2 upregulated IL-4 promoter activity in mast cells in a process, which required NFAT binding site and was sensitive to calcineurin (CaN) inhibitors. Furthermore Hg2+ activated transcription driven by artificial NFAT-dependent promoter containing three NFAT sites and increased CaN activity in vitro. These observations suggest that Hg2+ ions increase activity of CaN that in turn upregulates NFAT, which binds to specific DNA motif present in IL-4 promoter resulting in IL-4 expression. Thus, Hg2+ ions are able to activate both JNK and calcineurin, the two signaling pathways in immune cells that control expression of several immunomodulatory cytokines, and this could be important molecular mechanism mediating immunotoxic activities of mercuric compounds observed in vivo.
We continue our effort to establish the role of 3?UTR of the IL-4 gene in mRNA destabilization. Our data indicate such a role that is a novel observation as there is not much known about posttranscriptional level of regulation of IL-4 expression. Another post-transcriptional mechanism regulating cytokine expression in mast cells studied in the laboratory is intracellular trafficking of TNF-(alpha) protein leading to the storage of this cytokine in mast cell granules. The transfection of mast cells with DNA constructs coding for the TNF-(alpha)-EGFP fusion protein resulted in the apparent granular pattern of fluorescence, which colocalised with several granule markers. We are now utilising this system to delineate the metabolic processes involved in TNF-(alpha) transport as well as to determine the minimal amino acid motifs, which are necessary for its direction into granules.
Some of topic of the Laboratory of Molecular Imunology research are continued in the Centre of Medical Biology of Polish Academy of Science in Lodz.
Novel technology for in vitro immunotoxi-city testing
Due to the new chemical safety regulation being now introduced in Europe around 30 000 chemicals will have to be tested to different extend for possible toxicity. This testing will be accomplished not earlier than in 2048, will require 12.8 milions of experimental animals, and is expected to cost 8,64 bilions euro. Therefore there is an increasing demand, for methods suitable for high-throughput screening that increase the speed and reduce the cost per chemical entity for safety evaluation. There are also obvious ethical reasons for implementing in vitro alternatives in safety evaluation in order to reduce, refine, and replace the use of laboratory animals for routine testing. The laboratory coordinated the collaborative effort of six research groups towards development of a new system for testing immunotoxic effects of xenobiotics in-vitro. We have developed a new system for testing immunotoxic effects of chemicals in vitro. Our patent pending technology called "Fluorescent Cell Chip" (FCC) does not involve experimental animals but instead is based on a number of immortalized genetically modified cell lines representing different phenotypes of cells regulating immune response in vivo. FCC consists of a panel of reporter cell lines that regulate the expression of a transgene coding for fluorescent protein in the same way as they regulate expression of cytokines. This test could be employed in a uniform high throughput system for screening tousends of compounds for their possible effect on expression of a number of cytokine genes, which is a well documented and valid endpoint for immunotoxicity. In testing of the prototype of FCC using chemicals with known biological activities 86% of immunotoxic compounds, including skin and pulmonary sensitizers, were positive.
Signaling pathways regulating cytokine expression in mast cells
The second topic of our research is regulation of cytokine expression in mast cells with special emphasis on signal transduction mechanisms engaged in xenobiotic-mediated upregulation of IL-4 and TNF-(alpha) expression. We observed that several metal and transition metal ions induced and enhanced allergen-mediated IL-4 expression. All these effects of metal and transition metal ions on mast cells were observed at concentrations, which might be relevant for the environmental exposure in air pollution.
We investigated the role of JNK in signal transduction mechanisms regulating IL-4 expression in mast cells exposed to mercuric ions. We have gained new evidences for the critical role of JNK in upregulation of IL-4 by antigen and mercuric ions. The employment of a specific JNK inhibitor SP600125 resulted in the complete inhibition of c-Jun phosphorylation and IL-4 secretion. The JNK inhibitor exercised such effect in mast cells activated with antigen or exposed to mercuric chloride or activated concomitantly with both stimuli. These results support the hypothesis that JNK may be a focal point, which allows for a cross talk of two signaling pathways responding to antigen or xenobiotics and regulating cytokine expression in immune cells. Continuing our search for molecular mechanism of mercury-mediated IL-4 expression we investigated effect of mercuric ions on IL-4 promoter activity in mast cells. HgCl2 upregulated IL-4 promoter activity in mast cells in a process, which required NFAT binding site and was sensitive to calcineurin (CaN) inhibitors. Furthermore Hg2+ activated transcription driven by artificial NFAT-dependent promoter containing three NFAT sites and increased CaN activity in vitro. These observations suggest that Hg2+ ions increase activity of CaN that in turn upregulates NFAT, which binds to specific DNA motif present in IL-4 promoter resulting in IL-4 expression. Thus, Hg2+ ions are able to activate both JNK and calcineurin, the two signaling pathways in immune cells that control expression of several immunomodulatory cytokines, and this could be important molecular mechanism mediating immunotoxic activities of mercuric compounds observed in vivo.
We continue our effort to establish the role of 3?UTR of the IL-4 gene in mRNA destabilization. Our data indicate such a role that is a novel observation as there is not much known about posttranscriptional level of regulation of IL-4 expression. Another post-transcriptional mechanism regulating cytokine expression in mast cells studied in the laboratory is intracellular trafficking of TNF-(alpha) protein leading to the storage of this cytokine in mast cell granules. The transfection of mast cells with DNA constructs coding for the TNF-(alpha)-EGFP fusion protein resulted in the apparent granular pattern of fluorescence, which colocalised with several granule markers. We are now utilising this system to delineate the metabolic processes involved in TNF-(alpha) transport as well as to determine the minimal amino acid motifs, which are necessary for its direction into granules.
Some of topic of the Laboratory of Molecular Imunology research are continued in the Centre of Medical Biology of Polish Academy of Science in Lodz.
