Available online http://arthritis-research.com/content/7/1/R101 ResearchVol 7 No 1 article Open Access Gene expression profiles in the rat streptococcal cell wall-induced arthritis model identified using microarray analysis Inmaculada Rioja1, Chris L Clayton2, Simon J Graham2, Paul F Life1 and Marion C Dickson1 1Rheumatoid Arthritis Disease Biology Department, GlaxoSmithKline, Medicines Research Centre, Stevenage, UK 2Transcriptome Analysis Department, GlaxoSmithKline, Medicines Research Centre, Stevenage, UK Corresponding author: Inmaculada Rioja, [email protected] Received: 3 Jul 2004 Revisions requested: 16 Sep 2004 Revisions received: 4 Oct 2004 Accepted: 9 Oct 2004 Published: 19 Nov 2004 Arthritis Res Ther 2005, 7:R101-R117 (DOI 10.1186/ar1458)http://arthritis-research.com/content/7/1/R101 © 2004 Rioja et al., licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/ 2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is cited. Abstract Experimental arthritis models are considered valuable tools for P < 0.01), showing specific levels and patterns of gene delineating mechanisms of inflammation and autoimmune expression. The genes exhibiting the highest fold increase in phenomena. Use of microarray-based methods represents a expression on days -13.8, -13, or 3 were involved in chemotaxis, new and challenging approach that allows molecular dissection inflammatory response, cell adhesion and extracellular matrix of complex autoimmune diseases such as arthritis. In order to remodelling. Transcriptome analysis identified 10 upregulated characterize the temporal gene expression profile in joints from genes (Delta > 5), which have not previously been associated the reactivation model of streptococcal cell wall (SCW)-induced with arthritis pathology and are located in genomic regions arthritis in Lewis (LEW/N) rats, total RNA was extracted from associated with autoimmune disease. The majority of the ankle joints from naïve, SCW injected, or phosphate buffered downregulated genes were associated with metabolism, saline injected animals (time course study) and gene expression transport and regulation of muscle development. In conclusion, was analyzed using Affymetrix oligonucleotide microarray the present study describes the temporal expression of multiple technology (RAE230A). After normalization and statistical disease-associated genes with potential pathophysiological analysis of data, 631 differentially expressed genes were sorted roles in the reactivation model of SCW-induced arthritis in Lewis into clusters based on their levels and kinetics of expression (LEW/N) rat. These findings improve our understanding of the using Spotfire® profile search and K-mean cluster analysis. molecular events that underlie the pathology in this animal Microarray-based data for a subset of genes were validated model, which is potentially a valuable comparator to human using real-time PCR TaqMan® analysis. Analysis of the rheumatoid arthritis (RA). microarray data identified 631 genes (441 upregulated and 190 downregulated) that were differentially expressed (Delta > 1.8, Keywords: arthritis, differential gene expression, microarray, rat, SCW induced arthritis Introduction molecular complexity and pathogenesis of arthritis has Rheumatoid arthritis (RA) is an autoimmune chronic inflam- arisen from oligonucleotide-based microarray technology matory disease of unknown aetiology that is characterized [3], because this platform provides an opportunity to ana- by infiltration of monocytes, T cells and polymorphonuclear lyze simultaneously the expression of a large number of cells into the synovial joints. The pathogenesis of this dis- genes in disease tissues. ease is still poorly understood, and fundamental questions regarding the precise molecular nature and biological sig- The earliest preclinical stages of human RA are not easily nificance of the inflammatory changes remain to be accessible to investigation, but a diverse range of experi- answered [1,2]. A powerful way to gain insight into the mental arthritis models are considered valuable tools for ANOVA = analysis of variance; CCL = CC chemokine ligand; CCR = CC chemokine receptor; CXCL = CXC chemokine ligand; CXCR = CXC chem- okine receptor; ECM = extracellular matrix; EST = expressed sequence tag; IL = interleukin; MCP = monocyte chemoattractant protein; MHC = major histocompatibility complex; MIP = macrophage inflammatory protein; MMP = matrix metalloproteinase; NF-κB = nuclear factor-κB; NK = natural killer; NOS = nitric oxide synthase; PBS = phosphate-buffered saline; PCA = principal component analysis; PCR = polymerase chain reaction; PG-PS = peptidoglycan–polysaccharide; QTL = quantitative trait locus; RA = rheumatoid arthritis; RT = reverse transcription; SCW = streptococcal cell wall; SLPI = secretory leucocyte protease inhibitor; TIMP = tissue inhibitor of matrix metalloproteinase; TNF = tumour necrosis factor. R101 Arthritis Research & Therapy Vol 7 No 1 Rioja et al. delineating mechanisms of inflammation and autoimmune which provides a highly accurate method for quantifying phenomena. An animal model that shares some of the hall- mRNA expression levels for any particular differentially marks of human RA is the reactivation model of streptococ- expressed gene. To further investigate the possible associ- cal cell wall (SCW)-induced arthritis in rats. In this model, a ation of 20 selected upregulated genes with arthritis patho- synovitis with maximal swelling at 24 hours is induced by genesis, their chromosomal locations and the local injection of SCW antigen directly into an ankle joint. chromosomal locations of their corresponding human The initial response is reactivated by systemic (intravenous) orthologue were compared with the locations of previously challenge with SCW, which produces a more prolonged reported quantitative trait loci (QTLs) for inflammation, and severe inflammation confined to the joint previously arthritis and other autoimmune diseases. Our findings injected with SCW. In contrast to some other animal mod- show, for the first time, the gene expression profiles and els, in which the arthritic response develops gradually and kinetics of expression of hundreds of genes that are differ- unpredictably, in this model the flare response develops entially expressed in arthritic joints from the reactivation synchronously, allowing precise analysis of pathophysio- model of SCW-induced arthritis in Lewis (LEW/N) rat, logical mechanisms [4,5]. thereby improving our understanding of the biological path- ways that contribute to the pathogenesis of arthritis in this Some pathological changes observed in SCW-induced animal model and providing a valuable comparator to arthritis that are of relevance to human RA include infiltra- human RA. tion of polymorphonuclear cells, CD4+ T cells and macro- phages, hyperplasia of the synovial lining layer, pannus Methods formation and moderate erosion of cartilage and bone [4]. Reagents Previous reports have shown the dependency of this model The peptidoglycan–polysaccharide (PG-PS) 100p fraction on tumour necrosis factor (TNF)-α, IL-1α, IL-4, P-selectin, of SCW was purchased from Lee Laboratories (Grayson, vascular cell adhesion molecule-1, macrophage inflamma- GA, USA). RAE230A Affymetrix GeneChip® were pur- tory protein (MIP)-2, MIP-1α and monocyte chemoattract- chased from Affymetrix Inc. All reagents required for RT- ant protein (MCP)-1 [6,7]. Although the involvement of PCR were from PE Applied Biosystems (Warrington, UK). nitric oxide synthase (NOS) [8] and cyclo-oxygenase [9] in Forward and reverse primers were purchased from Invitro- the development of SCW-induced arthritis has also been gen™ Life Technologies (Invitrogen Ltd, Paisley, UK). Taq- noted, a global analysis of coordinated gene expression Man® probes were synthesized by PE Applied Biosystems. during the time course of disease in this experimental arthri- RiboGreen, used to quantify RNA, was obtained from tis model has not been investigated. Molecular Probes Inc. (Leiden, The Netherlands) and RNA 6000 Nano LabChip Kit®, used to assess RNA integrity, Arthritis involves many cell types from tissues adjacent to was from Agilent Technologies Inc. (Stockport, UK). the synovium. Therefore, as shown in previous studies [10,11], analysis of gene expression profiles by processing Animals whole homogenized joints can provide useful information All in vivo studies were undertaken in certified, dedicated about dysregulated genes, not only in synoviocytes but also in vivo experimental laboratories at the GlaxoSmithKline in other, neighbouring cells (myocytes, osteocytes and Medicines Research Centre (Stevenage, UK). The studies chondrocytes) that may also contribute to disease complied with national legislation and with local policies on pathology. the care and use of animals, and with related codes of prac- tice. Male Lewis (LEW/N) rats obtained from Harlan UK Ltd In the present study, whole homogenized rat ankle joints (Oxon, UK), at age 6–7 weeks, were housed under stand- from naïve, SCW-injected and phosphate-buffered saline ard conditions and received food and water ad libitum. Ani- (PBS; vehicle)-injected animals, included in a time-course mals were habituated to the holding room for a minimum of study, were analyzed for differential gene expression using 1 week before the experimental procedures.