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Identification of novel monosodium urate crystal regulated mRNAs by transcript profiling of dissected murine air pouch membranes.

Pessler F, Mayer CT, Jung SM, Behrens EM, Dai L, Menetski JP, Schumacher HR - Arthritis Res. Ther. (2008)

Bottom Line: The other mRNAs rose up to 200-fold within the subsequent 3 to 8 hours.The marked rise of the upregulated mRNAs after the early surge in cytokine and Egr-1 mRNAs suggests that they may be part of a 'second wave' of factors that amplify or perpetuate inflammation.Transcript profiling of the isolated air pouch membrane promises to be a powerful tool for identifying genes that act at different stages of inflammation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Klinik und Poliklinik für Kinder und Jugendmedizin, Technische Universität Dresden, Fetscherstrasse, 01307 Dresden, Germany. Frank.pessler@uniklinikum-dresden.de

ABSTRACT

Introduction: The murine air pouch is a bursa-like space that resembles the human synovial membrane. Injection of monosodium urate (MSU) crystals into the pouch elicits an acute inflammatory response similar to human gout. We conducted the present study to identify mRNAs that were highly regulated by MSU crystals in the pouch membrane.

Methods: Air pouch membranes were meticulously dissected away from the overlying skin. Gene expression differences between MSU crystal stimulated and control membranes were determined by oligonucleotide microarray analysis 9 hours after injection of MSU crystals or buffer only. Differential regulation of selected targets was validated by relative quantitative PCR in time course experiments with dissected air pouch membranes and murine peritoneal macrophages.

Results: Eleven of the 12 most highly upregulated mRNAs were related to innate immunity and inflammation. They included mRNAs encoding histidine decarboxylase (the enzyme that synthesizes histamine), IL-6, the cell surface receptors PUMA-g and TREM-1, and the polypeptides Irg1 and PROK-2. IL-6 mRNA rose 108-fold 1 hour after crystal injection, coinciding with a surge in mRNAs encoding IL-1beta, tumour necrosis factor-alpha and the immediate early transcription factor Egr-1. The other mRNAs rose up to 200-fold within the subsequent 3 to 8 hours. MSU crystals induced these mRNAs in a dose-dependent manner in cultured macrophages, with similar kinetics but lower fold changes. Among the downregulated mRNAs, quantitative PCR confirmed significant decreases in mRNAs encoding TREM-2 (an inhibitor of macrophage activation) and granzyme D (a constituent of natural killer and cytotoxic T cells) within 50 hours after crystal injection.

Conclusion: This analysis identified several genes that were previously not implicated in MSU crystal inflammation. The marked rise of the upregulated mRNAs after the early surge in cytokine and Egr-1 mRNAs suggests that they may be part of a 'second wave' of factors that amplify or perpetuate inflammation. Transcript profiling of the isolated air pouch membrane promises to be a powerful tool for identifying genes that act at different stages of inflammation.

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MSU crystal dose response. Mouse peritoneal macrophages were grown in medium overnight. After removal of nonadherent cells, medium containing increasing concentrations of monosodium urate (MSU) crystals was added. RNA was harvested 4 hours after the addition of crystal-containing medium and analyzed for target gene expression by TaqMan real-time reverse transcription PCR. Results represent the averages of two experiments.
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Figure 6: MSU crystal dose response. Mouse peritoneal macrophages were grown in medium overnight. After removal of nonadherent cells, medium containing increasing concentrations of monosodium urate (MSU) crystals was added. RNA was harvested 4 hours after the addition of crystal-containing medium and analyzed for target gene expression by TaqMan real-time reverse transcription PCR. Results represent the averages of two experiments.

Mentions: The induction of IL-1β, IL-6, TREM-1, PUMA-g, Hdc, PROK-2 and Irg1 by MSU crystals was also quantified in a dose response experiment (Figure 6). IL-1β and IL-6 mRNAs were induced maximally at a crystal concentration of 375 μg/ml medium, as were PUMA-g, Hdc and PROK-2 mRNAs. Induction of TREM-1 mRNA peaked at a slightly lower, and that of Irg1 mRNA at the lowest crystal concentration.


Identification of novel monosodium urate crystal regulated mRNAs by transcript profiling of dissected murine air pouch membranes.

Pessler F, Mayer CT, Jung SM, Behrens EM, Dai L, Menetski JP, Schumacher HR - Arthritis Res. Ther. (2008)

MSU crystal dose response. Mouse peritoneal macrophages were grown in medium overnight. After removal of nonadherent cells, medium containing increasing concentrations of monosodium urate (MSU) crystals was added. RNA was harvested 4 hours after the addition of crystal-containing medium and analyzed for target gene expression by TaqMan real-time reverse transcription PCR. Results represent the averages of two experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2483455&req=5

Figure 6: MSU crystal dose response. Mouse peritoneal macrophages were grown in medium overnight. After removal of nonadherent cells, medium containing increasing concentrations of monosodium urate (MSU) crystals was added. RNA was harvested 4 hours after the addition of crystal-containing medium and analyzed for target gene expression by TaqMan real-time reverse transcription PCR. Results represent the averages of two experiments.
Mentions: The induction of IL-1β, IL-6, TREM-1, PUMA-g, Hdc, PROK-2 and Irg1 by MSU crystals was also quantified in a dose response experiment (Figure 6). IL-1β and IL-6 mRNAs were induced maximally at a crystal concentration of 375 μg/ml medium, as were PUMA-g, Hdc and PROK-2 mRNAs. Induction of TREM-1 mRNA peaked at a slightly lower, and that of Irg1 mRNA at the lowest crystal concentration.

Bottom Line: The other mRNAs rose up to 200-fold within the subsequent 3 to 8 hours.The marked rise of the upregulated mRNAs after the early surge in cytokine and Egr-1 mRNAs suggests that they may be part of a 'second wave' of factors that amplify or perpetuate inflammation.Transcript profiling of the isolated air pouch membrane promises to be a powerful tool for identifying genes that act at different stages of inflammation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Klinik und Poliklinik für Kinder und Jugendmedizin, Technische Universität Dresden, Fetscherstrasse, 01307 Dresden, Germany. Frank.pessler@uniklinikum-dresden.de

ABSTRACT

Introduction: The murine air pouch is a bursa-like space that resembles the human synovial membrane. Injection of monosodium urate (MSU) crystals into the pouch elicits an acute inflammatory response similar to human gout. We conducted the present study to identify mRNAs that were highly regulated by MSU crystals in the pouch membrane.

Methods: Air pouch membranes were meticulously dissected away from the overlying skin. Gene expression differences between MSU crystal stimulated and control membranes were determined by oligonucleotide microarray analysis 9 hours after injection of MSU crystals or buffer only. Differential regulation of selected targets was validated by relative quantitative PCR in time course experiments with dissected air pouch membranes and murine peritoneal macrophages.

Results: Eleven of the 12 most highly upregulated mRNAs were related to innate immunity and inflammation. They included mRNAs encoding histidine decarboxylase (the enzyme that synthesizes histamine), IL-6, the cell surface receptors PUMA-g and TREM-1, and the polypeptides Irg1 and PROK-2. IL-6 mRNA rose 108-fold 1 hour after crystal injection, coinciding with a surge in mRNAs encoding IL-1beta, tumour necrosis factor-alpha and the immediate early transcription factor Egr-1. The other mRNAs rose up to 200-fold within the subsequent 3 to 8 hours. MSU crystals induced these mRNAs in a dose-dependent manner in cultured macrophages, with similar kinetics but lower fold changes. Among the downregulated mRNAs, quantitative PCR confirmed significant decreases in mRNAs encoding TREM-2 (an inhibitor of macrophage activation) and granzyme D (a constituent of natural killer and cytotoxic T cells) within 50 hours after crystal injection.

Conclusion: This analysis identified several genes that were previously not implicated in MSU crystal inflammation. The marked rise of the upregulated mRNAs after the early surge in cytokine and Egr-1 mRNAs suggests that they may be part of a 'second wave' of factors that amplify or perpetuate inflammation. Transcript profiling of the isolated air pouch membrane promises to be a powerful tool for identifying genes that act at different stages of inflammation.

Show MeSH
Related in: MedlinePlus