Research article

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

Frank Pessler^1,2 , Christian T Mayer^3,4 , Sung Mun Jung^4,5 , Ed M Behrens¹ , Lie Dai^4,6 , Joseph P Menetski⁷ and H Ralph Schumacher^4,8

¹Klinik und Poliklinik für Kinder und Jugendmedizin, Technische Universität Dresden, Fetscherstraße, 01307 Dresden, Germany

²Division of Rheumatology, The Children's Hospital of Philadelphia, Civic Center Blvd, Philadelphia, Pennsylvania 19104, USA

³Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universität München, Trogerstraße, 81675 München, Germany

⁴Division of Rheumatology, University of Pennsylvania, Spruce St, Philadelphia, Pennsylvania 19104, USA

⁵Faculty of Oriental Medicine, Department of Herbal Pharmacology, Kyung Hee University College of Oriental Medicine, Hoekidong, Dongdaemoonku, Seoul 130-701, Korea

⁶Division of Rheumatology, Second Affiliated Hospital, Sun Yat-sen University, Yan Jiang West Road, Guangzhou 510120, PR China

⁷Merck Research Laboratories, E. Lincoln Avenue, PO Box 2000, Rahway, New Jersey 07065, USA

⁸Division of Rheumatology, Veteran Affairs Medical Center, University and Woodland Avenues, Philadelphia, Pennsylvania 19104, USA

author email corresponding author email

Arthritis Research & Therapy 2008, 10:R64doi:10.1186/ar2435

Published:	3 June 2008

See related editorial by So, http://arthritis-research.com/content/10/3/112

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-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.