Evaluation of glyceraldehyde-3-phosphate, prolylpeptidyl isomerase A, and a set of stably expressed genes as reference mRNAs in urate crystal inflammation

ABSTRACT

V(D)J recombination in mouse thymocytes: Double-strand breaks near T cell receptor δ rearrangement signals

In the murine T cell receptor δ locus, V(D)J recombination events frequently involve the D2 and J1 elements. Here we report the presence of double-strand breaks at recombination signals flanking D2 in approximately 2% of thymus DNA. An excised linear species containing the sequences between D2 and J1 and a circular product of the joining of D2 and J1 recombination signals were also found. Although broken molecules with signal ends were detected, no species with coding ends could be identified. Observation of these broken molecules in thymus, but not in liver or spleen, provides the first direct evidence for an association between specific cleavage of chromosomal DNA and recombination in mammalian cells, and supports a breakage-reunion model of V(D)J recombination

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

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-1β, tumour necrosis factor-α 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

Validating differential regulation of mRNAs during MSU crystal inflammation in dissected air pouch membranes

Results were obtained from dissected membranes from the pouches used for the time course shown in Figure 1b. Negative control pouches were injected with 1 ml phosphate-buffered saline (PBS) and dissected at 9 hours. A, C and D: RNA was analyzed with TaqMan real-time reverse transcription PCR for the targets indicated. The legend text also shows values for mean fold expression changes that were measured at 9 hours (relative to 0 hours) in monosodium urate (MSU) crystal stimulated versus PBS injected (control) membranes. mRNA quantification of tumour necrosis factor (TNF)-α (MSU:PBS at 9 h = 14.1:1.7), interleukin (IL)-6 (MSU:PBS = 12.9:0.5), IL-1β (MSU:PBS = 34.7:0.7), and early growth response (Egr)-1 (MSU:PBS = 3.7:1.4). Left: determination of IL-6 protein concentration at 9 hours in the pouch exudate from pouches injected with PBS or MSU crystals (immunoassay). Center and right: immunohistochemical detection of IL-6 in the air pouch membrane. Chromogen: DAB (brown). Center: striated muscle (m) showing specific IL-6 immunostain; hair follicles (f) with nonspecific immunostain that was also seen with control immunoglobulin (original magnification, 50×). Right: specific IL-6 immunostaining in the inflamed pouch membrane (original magnification, 400×). mRNA quantification of triggering receptor expressed on myeloid cells (TREM)-1 (MSU:PBS at 9 h = 15.3:0.6), immunoresponsive gene (Irg)1 (MSU:PBS, 65:1.0), prokineticin (PROK)-2 (MSU:PBS = 58.4:1.0), histidine decarboxylase (Hdc; MSU:PBS = 60.4:1.3), and protein upregulated on macrophages activated with interferon-γ (PUMA-g; MSU:PBS = 120:1.3). mRNA quantification of TREM-2 (MSU:PBS at 9 h = 0.2:0.9), granzyme D (MSU:PBS = 0.5:0.8), leukemia/lymphoma-related factor (LRF; MSU:PBS = 1.8:1.7), and Nab2 (MSU:PBS = 0.8:0.9).<p><b>Copyright information:</b></p><p>Taken from "Identification of novel monosodium urate crystal regulated mRNAs by transcript profiling of dissected murine air pouch membranes"</p><p>http://arthritis-research.com/content/10/3/R64</p><p>Arthritis Research & Therapy 2008;10(3):R64-R64.</p><p>Published online 3 Jun 2008</p><p>PMCID:PMC2483455.</p><p></p

Validating differential regulation of mRNAs during MSU crystal inflammation in mouse peritoneal macrophages

Cells were harvested at the indicated time points after the addition of medium containing monosodium urate (MSU) crystals (200 μg/ml) or medium alone. RNA was analyzed by TaqMan real-time reverse transcription PCR for expression of the targets indicated in the figure. Results represent the averages of two experiments. Induction of target mRNAs in negative control (medium only) cells was negligible in nearly all cases. Therefore, the curves corresponding to the negative controls are not shown. However, numeric values for mean fold expression changes in MSU stimulated and negative controls with respect to t = 0 hours are listed below for the time points of maximal induction by MSU crystals. Tumour necrosis factor (TNF)-α (2 hours: MSU:medium = 28.1:1.2), IL-6 (1 hour: MSU:medium = 49.7:1.2), IL-1β (2 hours: MSU:medium = 13.1:0.5); and early growth response (Egr)-1 (1 hour: MSU:medium = 17.4:1). Irg1 (6 hours: MSU:medium = 60.0:6.8); prokineticin (PROK)-2 (6 hours: MSU:medium, 11.0:1.0), histidine decarboxylase (Hdc; 9 hours: MSU:medium = 11.6:0.9) and protein upregulated on macrophages activated with interferon-γ (PUMA-g; 9 hours: MSU:medium = 73.5:2.5). Comparison of fold induction by MSU crystals in dissected membranes versus macrophage culture.<p><b>Copyright information:</b></p><p>Taken from "Identification of novel monosodium urate crystal regulated mRNAs by transcript profiling of dissected murine air pouch membranes"</p><p>http://arthritis-research.com/content/10/3/R64</p><p>Arthritis Research & Therapy 2008;10(3):R64-R64.</p><p>Published online 3 Jun 2008</p><p>PMCID:PMC2483455.</p><p></p