Abstract
IntroductionThe 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.MethodsAir 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.ResultsEleven 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.ConclusionThis 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.
Highlights
The murine air pouch is a bursa-like space that resembles the human synovial membrane
This analysis identified several genes that were previously not implicated in monosodium urate (MSU) crystal inflammation
CSF = colony-stimulating factor; Egr = early growth response; Hdc = histidine decarboxylase; IL = interleukin; Irg = immunoresponsive gene; LRF = leukaemia/lymphoma-related factor; MSU = monosodium urate; NK = natural killer; PBS = phosphate-buffered saline; PCR = polymerase chain reaction; PROK = prokineticin; PUMA-g = protein upregulated on macrophages activated with interferon-γ; qPCR = quantitative polymerase chain reaction; TLR = Toll-like receptor; TNF = tumour necrosis factor; TREM = triggering receptor expressed on myeloid cells
Summary
The murine air pouch is a bursa-like space that resembles the human synovial membrane. Within several days of injecting a small volume of air (2 to 3 ml), a membrane of several layers of cells, which consist mostly of fibroblasts, mononuclear cells and small blood vessels, grows around this air-filled space [1] This membrane resembles the synovial membrane histologically and has important properties of the synovial lining, such as hyaluronic acid synthesis [2] and expression of the Ia antigen [1]. Microarray analysis of dissected inflamed and control membranes, coupled with validation of differential expression by relative quantitative polymerase chain reaction (qPCR), revealed a high yield of genes involved in innate immune responses and identified highly inducible mRNAs that were previously not implicated in crystal-induced inflammation
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