Figure 4.

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. (a) 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). (b) 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×). (c) 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). (d) 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).