Limits...
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.

Show MeSH

Related in: MedlinePlus

Histologic and molecular characterization of dissected membranes and adjacent tissues (a) Histologic cross-sections illustrating the plane of dissection. Left image: cross-section through an entire monosodium urate (MSU) crystal inflamed pouch wall, showing membrane (short red arrow) and the overlying cutaneous soft tissue (long black arrow). Original magnification: 100×. Center image: cutaneous soft tissue of the air pouch wall after removal of the membrane by the dissection method outlined in Figure 2. Original magnification: 100×. Right image: normal dorsal skin. It is nearly identical in appearance to the cutaneous parts shown in part b, which are left after dissection of the membrane. Original magnification: 100×. (b) Tissues that will probably contaminate the dissected membrane if they are not avoided during the final steps of the dissection. Left image: tissue from the nuchal cape-like structure to which the most rostral parts of the membrane are often attached. Original magnification: 200×. Center image: tissue obtained from the paraspinal ridges from which the base of the membrane arises (for the macroscopic appearance see the tissue marked with the arrow in Figure 2k). Original magnification: 200×. Right image: dissected membrane obtained from an air pouch injected with MSU crystals (2 mg in 1 ml phosphate-buffered saline). It consists mostly of fibroblasts and inflammatory cells. Blood vessels can also be found but are not abundant. Original magnification: 100×. (c) Partitioning of selected mRNAs between pouch membrane and the overlying cutaneous soft tissues. Membranes (n = 4) were dissected from the soft tissues 9 hours after injecting MSU crystals into the air pouches. RNA was extracted from dissected membranes or the soft tissues and analyzed separately by quantitative PCR. Results were normalized to GAPDH and the data obtained from membrane RNA arbitrarily assigned the value 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2483455&req=5

Figure 3: Histologic and molecular characterization of dissected membranes and adjacent tissues (a) Histologic cross-sections illustrating the plane of dissection. Left image: cross-section through an entire monosodium urate (MSU) crystal inflamed pouch wall, showing membrane (short red arrow) and the overlying cutaneous soft tissue (long black arrow). Original magnification: 100×. Center image: cutaneous soft tissue of the air pouch wall after removal of the membrane by the dissection method outlined in Figure 2. Original magnification: 100×. Right image: normal dorsal skin. It is nearly identical in appearance to the cutaneous parts shown in part b, which are left after dissection of the membrane. Original magnification: 100×. (b) Tissues that will probably contaminate the dissected membrane if they are not avoided during the final steps of the dissection. Left image: tissue from the nuchal cape-like structure to which the most rostral parts of the membrane are often attached. Original magnification: 200×. Center image: tissue obtained from the paraspinal ridges from which the base of the membrane arises (for the macroscopic appearance see the tissue marked with the arrow in Figure 2k). Original magnification: 200×. Right image: dissected membrane obtained from an air pouch injected with MSU crystals (2 mg in 1 ml phosphate-buffered saline). It consists mostly of fibroblasts and inflammatory cells. Blood vessels can also be found but are not abundant. Original magnification: 100×. (c) Partitioning of selected mRNAs between pouch membrane and the overlying cutaneous soft tissues. Membranes (n = 4) were dissected from the soft tissues 9 hours after injecting MSU crystals into the air pouches. RNA was extracted from dissected membranes or the soft tissues and analyzed separately by quantitative PCR. Results were normalized to GAPDH and the data obtained from membrane RNA arbitrarily assigned the value 1.

Mentions: Crucial steps in the dissection procedure are shown in Figure 2 and are also described in the Materials and methods section (see above). Dissected pouch membranes had a gelatinous but also somewhat fibrous consistency and usually weighed 70 to 110 mg. Membranes from MSU-stimulated pouches tended to be firmer and to rupture somewhat less easily during the dissection, probably because of a mild increase in thickness from inflammation [12]. Figure 3a illustrates the plane of dissection between the membrane and the overlying subcutaneous tissue. According to our observations, the air pouch membrane originates from longitudinal soft tissue ridges that overlie the paraspinal musculature and from a cape-like, thicker membrane in the nuchal area. Because pieces of these tissues might contaminate the membrane during the dissection and confound a gene expression analysis, we evaluated them histologically (Figure 3b). The nuchal structure was identified as adipose tissue (Figure 3b, left image) and thus originates from the nuchal fat pad. The paraspinal ridge tissue (shown macroscopically in Fig. 2, panel k) turned out to be rich in blood vessels, striated muscle and fascia (Figure 3b, centre) and thus probably was contiguous with the paraspinal muscles. To test the histologic purity of the dissected membranes, haematoxylin and eosin stains were prepared from several membranes. Adipocytes, skeletal muscle and fascia were not observed, confirming that the membranes had been dissected relatively free from surrounding tissue (Figure 3b, right).


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)

Histologic and molecular characterization of dissected membranes and adjacent tissues (a) Histologic cross-sections illustrating the plane of dissection. Left image: cross-section through an entire monosodium urate (MSU) crystal inflamed pouch wall, showing membrane (short red arrow) and the overlying cutaneous soft tissue (long black arrow). Original magnification: 100×. Center image: cutaneous soft tissue of the air pouch wall after removal of the membrane by the dissection method outlined in Figure 2. Original magnification: 100×. Right image: normal dorsal skin. It is nearly identical in appearance to the cutaneous parts shown in part b, which are left after dissection of the membrane. Original magnification: 100×. (b) Tissues that will probably contaminate the dissected membrane if they are not avoided during the final steps of the dissection. Left image: tissue from the nuchal cape-like structure to which the most rostral parts of the membrane are often attached. Original magnification: 200×. Center image: tissue obtained from the paraspinal ridges from which the base of the membrane arises (for the macroscopic appearance see the tissue marked with the arrow in Figure 2k). Original magnification: 200×. Right image: dissected membrane obtained from an air pouch injected with MSU crystals (2 mg in 1 ml phosphate-buffered saline). It consists mostly of fibroblasts and inflammatory cells. Blood vessels can also be found but are not abundant. Original magnification: 100×. (c) Partitioning of selected mRNAs between pouch membrane and the overlying cutaneous soft tissues. Membranes (n = 4) were dissected from the soft tissues 9 hours after injecting MSU crystals into the air pouches. RNA was extracted from dissected membranes or the soft tissues and analyzed separately by quantitative PCR. Results were normalized to GAPDH and the data obtained from membrane RNA arbitrarily assigned the value 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Histologic and molecular characterization of dissected membranes and adjacent tissues (a) Histologic cross-sections illustrating the plane of dissection. Left image: cross-section through an entire monosodium urate (MSU) crystal inflamed pouch wall, showing membrane (short red arrow) and the overlying cutaneous soft tissue (long black arrow). Original magnification: 100×. Center image: cutaneous soft tissue of the air pouch wall after removal of the membrane by the dissection method outlined in Figure 2. Original magnification: 100×. Right image: normal dorsal skin. It is nearly identical in appearance to the cutaneous parts shown in part b, which are left after dissection of the membrane. Original magnification: 100×. (b) Tissues that will probably contaminate the dissected membrane if they are not avoided during the final steps of the dissection. Left image: tissue from the nuchal cape-like structure to which the most rostral parts of the membrane are often attached. Original magnification: 200×. Center image: tissue obtained from the paraspinal ridges from which the base of the membrane arises (for the macroscopic appearance see the tissue marked with the arrow in Figure 2k). Original magnification: 200×. Right image: dissected membrane obtained from an air pouch injected with MSU crystals (2 mg in 1 ml phosphate-buffered saline). It consists mostly of fibroblasts and inflammatory cells. Blood vessels can also be found but are not abundant. Original magnification: 100×. (c) Partitioning of selected mRNAs between pouch membrane and the overlying cutaneous soft tissues. Membranes (n = 4) were dissected from the soft tissues 9 hours after injecting MSU crystals into the air pouches. RNA was extracted from dissected membranes or the soft tissues and analyzed separately by quantitative PCR. Results were normalized to GAPDH and the data obtained from membrane RNA arbitrarily assigned the value 1.
Mentions: Crucial steps in the dissection procedure are shown in Figure 2 and are also described in the Materials and methods section (see above). Dissected pouch membranes had a gelatinous but also somewhat fibrous consistency and usually weighed 70 to 110 mg. Membranes from MSU-stimulated pouches tended to be firmer and to rupture somewhat less easily during the dissection, probably because of a mild increase in thickness from inflammation [12]. Figure 3a illustrates the plane of dissection between the membrane and the overlying subcutaneous tissue. According to our observations, the air pouch membrane originates from longitudinal soft tissue ridges that overlie the paraspinal musculature and from a cape-like, thicker membrane in the nuchal area. Because pieces of these tissues might contaminate the membrane during the dissection and confound a gene expression analysis, we evaluated them histologically (Figure 3b). The nuchal structure was identified as adipose tissue (Figure 3b, left image) and thus originates from the nuchal fat pad. The paraspinal ridge tissue (shown macroscopically in Fig. 2, panel k) turned out to be rich in blood vessels, striated muscle and fascia (Figure 3b, centre) and thus probably was contiguous with the paraspinal muscles. To test the histologic purity of the dissected membranes, haematoxylin and eosin stains were prepared from several membranes. Adipocytes, skeletal muscle and fascia were not observed, confirming that the membranes had been dissected relatively free from surrounding tissue (Figure 3b, right).

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