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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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Related in: MedlinePlus

Outline of the air pouch experiments. (a) Sequence of events. Air is injected subcutaneously on day 0 and again on day 3 to keep the pouch inflated. On day 6 the remaining air is aspirated, and the monosodium urate (MSU) crystal suspension (2 mg in 1 ml phosphate-buffered saline [PBS]) or 1 ml PBS only is injected into the pouch cavity. Pouch exudate and tissue are obtained for analysis up to 50 hours after crystal injection. (b) Determination of the time of maximal inflammation in the pouch lumen. MSU crystal suspension (2 mg in 1 ml PBS) was injected into the pouch at t = 0 hours. Pouch exudate leukocyte counts were determined by manual cell counting at the indicated time points (n = 4 mice for each time point).
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Figure 1: Outline of the air pouch experiments. (a) Sequence of events. Air is injected subcutaneously on day 0 and again on day 3 to keep the pouch inflated. On day 6 the remaining air is aspirated, and the monosodium urate (MSU) crystal suspension (2 mg in 1 ml phosphate-buffered saline [PBS]) or 1 ml PBS only is injected into the pouch cavity. Pouch exudate and tissue are obtained for analysis up to 50 hours after crystal injection. (b) Determination of the time of maximal inflammation in the pouch lumen. MSU crystal suspension (2 mg in 1 ml PBS) was injected into the pouch at t = 0 hours. Pouch exudate leukocyte counts were determined by manual cell counting at the indicated time points (n = 4 mice for each time point).

Mentions: Figure 1a outlines the sequence of events of the air pouch experiments. Air pouches were raised on the backs of 6- to 8-week-old female BALB/c mice (Taconic, Tarrytown, NY, USA) by subcutaneous injection of 3 ml filtered air [1,7]. Pouches were re-inflated on day 3 with an additional 2 ml filtered air. MSU crystals were prepared in accordance with the method proposed by McCarty and Faires [8] and were determined to be free from endotoxin using the Gelclot LAL reagent (Charles River Labs, Wilmington, MA, USA). Aliquots from the same batch were used for all experiments. A suspension of 2 mg MSU crystals in 1 ml sterile endotoxin-free phosphate-buffered saline (PBS) was injected into the pouch on day 6. To verify the time points of peak and natural resolution of inflammation in the pouch, a 50-hour time course experiment was performed during which pouch exudate leukocyte counts were determined at several time points after injection of MSU crystals. In agreement with our previous findings [9], the leukocyte count rose 56-fold from 0 to 9 hours and then subsided, returning close to baseline by 50 hours (Figure 1b). Negative control pouches (n = 5) were injected with 1 ml PBS and harvested at 9 hours. Their mean leukocyte count was similar to that of the pouches at t = 0 hours (0.32 ± 0.18 × 106 cells/pouch at 9 hours versus 0.18 ± 0.09 at 0 hours; p = 0.31, one-tailed t-test). All animal experiments followed internationally recognized guidelines and were approved by the Institutional Animal Care and Research Committee of the Philadelphia VA Medical Center.


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)

Outline of the air pouch experiments. (a) Sequence of events. Air is injected subcutaneously on day 0 and again on day 3 to keep the pouch inflated. On day 6 the remaining air is aspirated, and the monosodium urate (MSU) crystal suspension (2 mg in 1 ml phosphate-buffered saline [PBS]) or 1 ml PBS only is injected into the pouch cavity. Pouch exudate and tissue are obtained for analysis up to 50 hours after crystal injection. (b) Determination of the time of maximal inflammation in the pouch lumen. MSU crystal suspension (2 mg in 1 ml PBS) was injected into the pouch at t = 0 hours. Pouch exudate leukocyte counts were determined by manual cell counting at the indicated time points (n = 4 mice for each time point).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Outline of the air pouch experiments. (a) Sequence of events. Air is injected subcutaneously on day 0 and again on day 3 to keep the pouch inflated. On day 6 the remaining air is aspirated, and the monosodium urate (MSU) crystal suspension (2 mg in 1 ml phosphate-buffered saline [PBS]) or 1 ml PBS only is injected into the pouch cavity. Pouch exudate and tissue are obtained for analysis up to 50 hours after crystal injection. (b) Determination of the time of maximal inflammation in the pouch lumen. MSU crystal suspension (2 mg in 1 ml PBS) was injected into the pouch at t = 0 hours. Pouch exudate leukocyte counts were determined by manual cell counting at the indicated time points (n = 4 mice for each time point).
Mentions: Figure 1a outlines the sequence of events of the air pouch experiments. Air pouches were raised on the backs of 6- to 8-week-old female BALB/c mice (Taconic, Tarrytown, NY, USA) by subcutaneous injection of 3 ml filtered air [1,7]. Pouches were re-inflated on day 3 with an additional 2 ml filtered air. MSU crystals were prepared in accordance with the method proposed by McCarty and Faires [8] and were determined to be free from endotoxin using the Gelclot LAL reagent (Charles River Labs, Wilmington, MA, USA). Aliquots from the same batch were used for all experiments. A suspension of 2 mg MSU crystals in 1 ml sterile endotoxin-free phosphate-buffered saline (PBS) was injected into the pouch on day 6. To verify the time points of peak and natural resolution of inflammation in the pouch, a 50-hour time course experiment was performed during which pouch exudate leukocyte counts were determined at several time points after injection of MSU crystals. In agreement with our previous findings [9], the leukocyte count rose 56-fold from 0 to 9 hours and then subsided, returning close to baseline by 50 hours (Figure 1b). Negative control pouches (n = 5) were injected with 1 ml PBS and harvested at 9 hours. Their mean leukocyte count was similar to that of the pouches at t = 0 hours (0.32 ± 0.18 × 106 cells/pouch at 9 hours versus 0.18 ± 0.09 at 0 hours; p = 0.31, one-tailed t-test). All animal experiments followed internationally recognized guidelines and were approved by the Institutional Animal Care and Research Committee of the Philadelphia VA Medical Center.

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