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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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Key steps in the separation of the membrane from the overlying soft tissues. (a) Mouse with dorsal air pouch just before dissection. (b) A small incision is made into the dorsal skin overlying the pouch. This incision is just deep enough to expose the apex of the pouch membrane. (c) The membrane is punctured with a scalpel or needle. (d) The pouch content is lavaged with 2 ml phosphate-buffered saline. If the opening is enlarged sufficiently, the lavage can be performed under direct visualization. (e-i) Using blunt dissection with curved clamps or curved scissors, the pouch membrane is separated meticulously from the overlying skin. The instrument follows a path of least resistance between the membrane and the overlying soft tissues. (j) Parts of the membrane adhering to the more caudal skin can be clipped off with fine scissors. Finally, the membrane collapses on the dorsum of the animal. (k, l) The membrane is then grasped with forceps, elevated and cut at the base with scissors. (m) The gelatinous appearing dissected membrane (arrow) adhering to the forceps just before homogenization in Trizol medium.
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Figure 2: Key steps in the separation of the membrane from the overlying soft tissues. (a) Mouse with dorsal air pouch just before dissection. (b) A small incision is made into the dorsal skin overlying the pouch. This incision is just deep enough to expose the apex of the pouch membrane. (c) The membrane is punctured with a scalpel or needle. (d) The pouch content is lavaged with 2 ml phosphate-buffered saline. If the opening is enlarged sufficiently, the lavage can be performed under direct visualization. (e-i) Using blunt dissection with curved clamps or curved scissors, the pouch membrane is separated meticulously from the overlying skin. The instrument follows a path of least resistance between the membrane and the overlying soft tissues. (j) Parts of the membrane adhering to the more caudal skin can be clipped off with fine scissors. Finally, the membrane collapses on the dorsum of the animal. (k, l) The membrane is then grasped with forceps, elevated and cut at the base with scissors. (m) The gelatinous appearing dissected membrane (arrow) adhering to the forceps just before homogenization in Trizol medium.

Mentions: Figure 2 shows key steps in the membrane dissection. After killing the animals by carbon dioxide asphyxiation, the apex of the pouch membrane was exposed with a small skin incision (panel b). The membrane was then punctured with a scalpel (panel c). Typically, little free exudate accumulates within the pouch. Leukocytes were therefore lavaged out of the pouch lumen with 2 ml PBS (panel d) and the leukocyte count in the resulting lavage fluid determined with a hemocytometer [10]. The pouch membrane was then separated meticulously from adjacent subcutaneous and paraspinal tissues by blunt dissection (panels e to j). Finally, the membrane was grasped with forceps, elevated and cut at the base (panels k and l). Great care was taken to avoid the paraspinal and nuchal tissues, to which the base of the membrane is typically attached. Using a rotatory homogenizer (Omni International, Warrenton, VA, USA), the isolated membranes were homogenized in Trizol medium (Invitrogen, Carlsbad, CA, USA), flash frozen in liquid nitrogen and stored at -70°C until further use.


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)

Key steps in the separation of the membrane from the overlying soft tissues. (a) Mouse with dorsal air pouch just before dissection. (b) A small incision is made into the dorsal skin overlying the pouch. This incision is just deep enough to expose the apex of the pouch membrane. (c) The membrane is punctured with a scalpel or needle. (d) The pouch content is lavaged with 2 ml phosphate-buffered saline. If the opening is enlarged sufficiently, the lavage can be performed under direct visualization. (e-i) Using blunt dissection with curved clamps or curved scissors, the pouch membrane is separated meticulously from the overlying skin. The instrument follows a path of least resistance between the membrane and the overlying soft tissues. (j) Parts of the membrane adhering to the more caudal skin can be clipped off with fine scissors. Finally, the membrane collapses on the dorsum of the animal. (k, l) The membrane is then grasped with forceps, elevated and cut at the base with scissors. (m) The gelatinous appearing dissected membrane (arrow) adhering to the forceps just before homogenization in Trizol medium.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Key steps in the separation of the membrane from the overlying soft tissues. (a) Mouse with dorsal air pouch just before dissection. (b) A small incision is made into the dorsal skin overlying the pouch. This incision is just deep enough to expose the apex of the pouch membrane. (c) The membrane is punctured with a scalpel or needle. (d) The pouch content is lavaged with 2 ml phosphate-buffered saline. If the opening is enlarged sufficiently, the lavage can be performed under direct visualization. (e-i) Using blunt dissection with curved clamps or curved scissors, the pouch membrane is separated meticulously from the overlying skin. The instrument follows a path of least resistance between the membrane and the overlying soft tissues. (j) Parts of the membrane adhering to the more caudal skin can be clipped off with fine scissors. Finally, the membrane collapses on the dorsum of the animal. (k, l) The membrane is then grasped with forceps, elevated and cut at the base with scissors. (m) The gelatinous appearing dissected membrane (arrow) adhering to the forceps just before homogenization in Trizol medium.
Mentions: Figure 2 shows key steps in the membrane dissection. After killing the animals by carbon dioxide asphyxiation, the apex of the pouch membrane was exposed with a small skin incision (panel b). The membrane was then punctured with a scalpel (panel c). Typically, little free exudate accumulates within the pouch. Leukocytes were therefore lavaged out of the pouch lumen with 2 ml PBS (panel d) and the leukocyte count in the resulting lavage fluid determined with a hemocytometer [10]. The pouch membrane was then separated meticulously from adjacent subcutaneous and paraspinal tissues by blunt dissection (panels e to j). Finally, the membrane was grasped with forceps, elevated and cut at the base (panels k and l). Great care was taken to avoid the paraspinal and nuchal tissues, to which the base of the membrane is typically attached. Using a rotatory homogenizer (Omni International, Warrenton, VA, USA), the isolated membranes were homogenized in Trizol medium (Invitrogen, Carlsbad, CA, USA), flash frozen in liquid nitrogen and stored at -70°C until further use.

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