Expression profiling of select cytokines in canine osteoarthritis tissues

Veterinary Immunology and Immunopathology 118 (2007) 59–67 www.elsevier.com/locate/vetimm

Expression profiling of select cytokines in canine osteoarthritis tissues Lindsey J. Maccoux a,b, Fiona Salway a, Philip J.R. Day a,b, Dylan N. Clements a,c,* a

Centre for Integrated Genomic Medical Research, University of Manchester, The Stopford Building, Oxford Road, Manchester, M13 9PT, UK b ISAS – Institute for Analytical Sciences, Bunsen-Kirchhoff-Str. 11, 44139 Dortmund, Germany c Musculoskeletal Diseases Research Group, Faculty of Veterinary Science, University of Liverpool, Liverpool, L69 3BX, UK Received 30 October 2006; received in revised form 23 March 2007; accepted 12 April 2007

Abstract The objective of this study was to investigate the level of expression of five cytokines in four different articular tissues from the joints of dogs with and without osteoarthritis (OA). Articular tissues were harvested from the stifle (fat, cranial cruciate ligament, synovial membrane) or hip (articular cartilage) from eight dogs with OA secondary to cranial cruciate ligament disease (stifle) or hip dysplasia (hip), undergoing routine surgical treatment for the condition, and from five dogs euthanatized without orthopaedic disease. The mRNA transcript numbers for interleukin-1b (IL-1b), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10) and interleukin-17 (IL-17) were determined by quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR). Increased expression of IL-1b, IL-6 and IL-10 in OA synovial membrane, increased expression of IL-1b and IL-6 in ruptured (OA) ligament, and reduced expression of IL-8 in OA synovial membrane were identified. Cytokine expression was detected in multiple tissues within the articular joint, but differential expression in OA was detected primarily in the synovial membrane and cranial cruciate ligament. # 2007 Elsevier B.V. All rights reserved. Keywords: Cytokine; Canine; Osteoarthritis; RT-qPCR

1. Introduction Osteoarthritis (OA) is a gradually progressing disorder of mammalian joints, characterised by the destruction of articular cartilage, which results in discomfort and dysfunction of the affected joint. OA is thought to originate from a series of complex interactions of both biochemical and biomechanical factors that occur simultaneously resulting in the

* Corresponding author. Tel.: +44 7803203551; fax: +44 1517944219. E-mail address: [email protected] (D.N. Clements). 0165-2427/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2007.04.006

breakdown of the cartilage matrix (Johnston, 1997). The biological and morphological changes initiated in OA are not constrained to articular cartilage, with synovial membrane (Dingle, 1981), infrapatella fat (Ushiyama et al., 2003), ligament (Hayashi et al., 2003) and bone (Brandt et al., 1991; Rogers et al., 2004) also being affected, although research in OA has concentrated predominately on the pathogenesis of articular cartilage destruction. The primary pathways responsible for the disturbance in the balance in degradation and repair of the cartilage matrix is believed to be mediated through cytokines and chemokines (Fernandes et al., 2002). Cytokine activities are associated with the functional alterations in synovial membrane, cartilage and subchondral bone and are

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produced spontaneously or subsequently stimulated by cells in joint tissue (Martel-Pelletier et al., 1999). In OA joints cytokines are hypothesized to originate primarily from the synovial membrane (Martel-Pelletier et al., 1999), which reflects the inflammatory changes recognised in this tissue (Bondeson et al., 2006). Many cytokines have been implicated in the pathogenesis of OA, such as IL-1a, IL-1b, IL-4, IL-6, IL-8, IL-10, IL-11, IL-13, IL-17, IL-18, leukaemia inhibitory factor (LIF) and tumor necrosis factor alpha (TNF-a) (MartelPelletier et al., 1999; Lotz, 2001; Fernandes et al., 2002; Goldring et al., 2004; Hegemann et al., 2005). For this study, a selection of cytokines suspected to have primary involvement in the development and progression of OA on the basis of previous in vitro and in vivo data were chosen for further investigation (IL-1b, IL-6, IL-8, IL-10, IL-17). Interleukin-1b is understood to be the cytokine that primarily drives OA, and inhibition of IL-1b abrogates macroscopic and histological severity of OA (Pelletier et al., 1997). IL-1b induces chondrocytes and synovial cells to produce catabolic proteases and stimulates the production of prostaglandin E2 and pro-inflammatory cytokines such as IL-6, IL-8 and LIF (Martel-Pelletier et al., 1999). IL-6 contributes to the pathological changes in OA at a cellular level by stimulating the proliferation of chondrocytes and increasing the number of neutrophils and monocytes in the synovial tissue (Guerne et al., 1989), and at a molecular level by inducing the amplification of IL-1 effects through the up-regulation of matrix metalloproteinase (MMP) synthesis and the inhibition of proteoglycan production (Nietfeld et al., 1990). IL-8 is a chemokine with modulatory activity, which stimulates neutrophil chemotaxis and IL-1, IL-6 and TNF-a release (Yu et al., 1994), although it can also be expressed by stimulated chondrocytes (Lotz et al., 1992). IL-17 is a pro-inflammatory cytokine which stimulates protease degradation and suppresses synthesis of proteoglycans in cartilage (Dudler et al., 2000; Chabaud et al., 2001). Additionally, IL-17 causes an increase in the production of other bio-active molecules such as IL-6 and IL-8 in cartilage, bone and synovial membrane (Moseley et al., 2003). Interleukin-10 is an anti-inflammatory cytokine, whose expression reduces pro-inflammatory cytokine (IL-1 and TNF-a) production in synovial membrane (Katsikis et al., 1994). The modulatory effects of IL-10 also extend to cartilage where IL-10 inhibits IL-1 induced MMP and nitric oxide production (Wang and Lou, 2001). The anti-inflammatory actions of IL-10 have made it a candidate for novel gene based treatment of OA (Zhang et al., 2004).

Little is known about the level of cytokine expression in the tissues of canine OA joints. Increased levels of IL1b and IL-6 have been detected in the synovial fluid of canine hips affected by hip dysplasia (Fujita et al., 2005), although in a similar study increased levels of IL6, but not IL-1b were detected in the synovial fluid of dogs with OA (Carter et al., 1999). The expression of IL-1b, IL-6, IL-8 and IL-10 have been recorded in the synovial fluid of dogs with OA (cranial cruciate ligament disease), albeit at lower levels than identified in the synovial fluid of dogs with rheumatoid arthritis (Hegemann et al., 2005). The primary goal of this study was to identify the relative expression of selected cytokines in different tissues from normal and OA canine joints. We hypothesized that the expression of selected cytokines (IL-1b, IL-6, IL-8, IL-17 and IL-10) would be changed in OA, and that the changes would be tissues specific. 2. Materials and methods 2.1. Sample collection and storage Infrapatellar fat (n = 6), ruptured cranial cruciate ligament (n = 5), and synovial membrane (n = 8) were obtained from dogs with clinical OA (as demonstrated by the presence of osteophytes on stifle radiographs) that was secondary to naturally occurring joint disease (cranial cruciate ligament rupture). Articular cartilage (n = 5) was removed from the femoral head of dogs with clinical OA of the hip secondary to naturally occurring joint disease (hip dysplasia). In each case the samples were obtained as part of the standard surgical treatment for the disease in question (total hip replacement or cranial cruciate ligament rupture surgery [tibial plateau levelling osteotomy]). Articular cartilage samples could not be harvested from matching stifle joints, as articular cartilage is not removed as part of the standard surgical procedure during cranial cruciate ligament rupture surgery. Furthermore it would not have been ethically acceptable to remove articular cartilage form these joints, as they were not undergoing an arthroplasty procedure. Consequently, articular cartilage samples were obtained from a different joint subject to an arthroplasty procedure (hip joints). Control samples (healthy) were obtained from the stifle joint (infrapatellar fat pad (n = 5), cranial cruciate ligament (n = 5) and synovial membrane (n = 5)) and hip joint (articular cartilage (n = 5)) of dogs euthanatized for reasons other than, and with no evidence of, joint disease. Following the collection of the tissue, the samples were weighed and immediately stored in RNAlaterTM (Qiagen Inc.,

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Crawley, UK), according to the manufacturers instructions, until extraction. 2.2. RNA extraction Total RNA was extracted using a phenol/guanidine hydrochloride reagent (Trizol, Invitrogen Ltd., UK) with a chloroform extraction and ethanol precipitation, for all of the tissue samples as previously described (Clements et al., 2006b). An on column DNA digestion step was included (RNase-Free DNase Set, Qiagen Ltd., Crawley, UK). Final elution of the total RNA was performed using 30 ml of RNase free water, and repeated to maximize the amount of RNA eluted. Total RNA samples were stored at 80 8C until use. The concentration of total RNA representing each sample was quantified by using a NanoDrop1 ND, 1000 UV/ visible Spectrophotometer (NanoDrop Technologies Ltd., Wilmington, Delaware, USA). 2.3. cDNA synthesis Reverse transcription was performed using Superscript II reverse transcriptase (Invitrogen, Dorset, UK) according to the manufacturers instructions (http:// www.invitrogen.com). Initially 200 mg (10 ml) total RNA was pre-incubated with 0.5 mg (1 ml) oligo-dT12– 18 (Invitrogen, Paisley, UK) and 10 mM (1 ml) dNTP mix (Invitrogen, Paisley, UK) at 658 for 5 min. After chilling on ice, 4 ml of 5 first strand buffer (containing 250 mM Tris–HCI (pH 8.3), 375 mM KC1, 15 mM MgCl2), 2 ml of 0.1 M DTT and 40 units (1 ml) of RNAsin (Promega, Southhampton, UK) were added to each sample and the samples incubated for 2 min at 42 8C, followed by the addition of 200 units (1 ml) of Superscript II reverse transcriptase (Invitrogen, Doreset, UK) and incubated for 50 min. Reverse transcriptase activity was terminated by incubation at 70 8C for 15 min, and samples stored at 80 8C until use. 2.4. Quantitative real-time reverse transcriptase PCR (RT-qPCR) assay design Transcript sequences for both reference genes and cytokines were obtained from the National Centre for Biotechnology Information (NCBI, http://www.ncbi. nlm.nih.gov/), and were cross referenced to the Ensembl canine genome database (www.ensembl.org). Primer and probe sequences were then designed for each of the reference genes and cytokines by using the Universal Probe Library Assay Design Centre (Roche Diagnostics Ltd., http://www.universalprobelibrary.com). BLAST

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searches were performed for all primer sequences to confirm gene specificity. Primers were synthesized by Metabion International AG (Martinsried, Germany), and probes were synthesized by Roche Diagnostics (Lewes, UK) using locked nucleic acid with 50 -end reporter dye fluorescein (FAM (6-carboxy fluorescein)) and 30 -end dark quencher dye. Template oligonucleotides (Mohammadi and Day, 2004) for the amplicon amplified by each assay were synthesized by Metabion (Martinsried, Germany). Novel reference genes were identified from microarray data (unpublished observations) and their stability measured using a standard analysis technique (Vandesompele et al., 2002). For each experiment, the two optimal reference genes were used, with gene expression stability measures and pair-wise variation recorded for each pair of reference gene (unpublished observations) well within the limits recommended by previous authors (Vandesompele et al., 2002). Primers and matched probes were selected for five reference genes; 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), CG14980PB (CG14980-PB), HIRA interacting protein 5 isoform 2 (HIRAP5), mitochondrial ribosomal protein S7 (S7), and mitochondrial 28S ribosomal protein S25 (MRPS25), and five cytokine genes; IL-1b, IL-6, IL-8, IL-10 and IL-17 (Table 1). For the normalisation of cytokine expression in articular tissues, the reference genes S7 and MRP-S25 were used for articular cartilage, S7 and IMP for synovial membrane, HIRAP5 and CG14980PB for cranial cruciate ligament, and CG14980-PB and S7 for infrapatellar fat. Quantitative real-time reverse transcriptase PCR assays were performed in triplicate using the Lightcycler1 480 (Roche Diagnostics, Lewes, UK) in 384 well format, with three no template controls used for each assay. The reaction volume in each well consisted of 5 mL LightCycler1 480 Probes Master 2 concentration (Roche Diagnostics) (containing FastStart Taq DNA polymerase, reaction buffer, dNTP mix (with dUTP) and 6.4 mM MgCl2), 0.7 mL of LightCycler1 480 Probes Master H2O (Roche Diagnostics), 0.1 mL of 20 mM forward primer, 0.1 mL of 20 mM reverse primer, 0.1 mL of 10 mM fluorescently-labelled probe and either 4 mL of sample cDNA, diluted template, or 4 mL of LightCycler1 480 Probes Master H2O. The standard amplification conditions consisted of 1 cycle at 95 8C for 5 min, followed by 50 cycles of 95 8C for 15 s and 60 8C for 30 s. Real-time data was then analysed by using Lightcycler1 480 Basic Software (Roche Diagnostics, Lewes, UK). Firstly, standard curves were generated for each assay with serial dilutions of the

62 Table 1 Primer and probe sequences, and the efficiency and error values determined from standard curves, for real-time PCR detection assays of cytokines and reference genes in different articular tissues Gene symbol NCBI accession number

Interleukin-1b

IL-1b

Interleukin-6

Forward (F) and reverse (R) primers

Probe sequence (number)

Selection PCR PCR of base efficiency error pairs values values

NM_001037971 F-TGCAAAACAGATGCGGATAA R-GTAACTTGCAGTCCACCGATT

GCAGCCAT (46)

365–28

IL-6

XM_850499

F-TGAACTCCCTCTCCACAAGC R-CGGGGTAGGGAAAGCAGTA

TGGTGATG (9)

Interleukin-8

IL-8

XM_850481

Interleukin-17

IL-17

Interleukin-10

IL-10

CG14980-PB

CG14980-PB XM_536878

Cut off Minimum Ct value number of transcripts detectable

1.874

0.0139 40.0

11 Molecules

59–129

1.678

0.144

0 Molecules

F-TGGGTACAAAAGGTTGTGCAG R-TTTGTTGTTTCACGGATCTTGT

AGGCTGAG (111 – Rat) 256–321

1.782

0.0927 40.0

476 Molecules

XM_538958

F-CACTCCTTCCGGCTAGAGAA R-CACATGGCGAACAATAGGG

GGTGGCTG (83)

556–627

2.018

0.0609 38.5

0 Molecules

XM_850467

F-CAGGTGAAGAGCGCATTTAGT R-TCAAACTCACTCATGGCTTTGT

GCTCCAGG (1)

425–489

1.862

0.0738 46.1

38 Molecules

F-GCAGGAAGGGATTCTCCAG GCCAGGAA (72) R-GGGTCCAGTAAGAAATCTTCCATAA

867–941

1.913

0.0462 38.25

0 Molecules 4 Molecules

38.7

5-Amino-imidazole-4IMP carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase

XM_858011

F-CGCTGCCTCTTTCAAACAT R-TTTGGCCTCATCTTCACTGAG

CAGCAGGT (62)

783–55

1.997

0.139

40.0

HIRA interacting protein HIRAP5 5 isoform 2

XM_850340

F-AATTCAGAACATGCTGCAATTTTA

AGGTGGAG (39)

666–735

1.903

0.0565 32.930

0 Molecules

Mitochondrial ribosomal protein S7

XM_846915

F-AGTGCAGGGAGAAGAAGCAC

GGATGCTG (89)

788–849

1.881

0.0309 40.0

1 Molecule

GCCAGGAA (72)

422–509

1.779

0.0382 40.0

59 Molecules

R-TGATTCATCATCCATAACCTGTTC S7

R-CAGCAGCTCGTGTGACAACT Mitochondrial 28S ribosomal protein S25

MRP-S25

XM_533729

F-TGAAGGTCATGACGGTGAAC R-TGGATCTGAGGTATGTTGAAAAAC

L.J. Maccoux et al. / Veterinary Immunology and Immunopathology 118 (2007) 59–67

Gene name

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template oligonucleotides. For all subsequent unknown assays a calibrator (dilution of the template oligonucleotide, of known template number) was included on the plate so that absolute quantities (transcript number) could be determined with reference to the standard curve. Absolute quantification was determined by the Lightcycler1 480 Basic software with reference to both the standard curve and template calibrator. As absolute quantification was performed with reference to the standard curve, assay efficiency did not need to be 100%. Serial dilutions of each amplicon were used to calculate the minimum number of transcript molecules which could be detected, and the associated threshold cycle values (Table 1). Data was then normalised by identifying the sample (control sample) with the lowest reference gene expression (average copy number of two most stably expressed reference genes) and had measurable expression in each of the cytokine assays. The other samples were normalised by dividing the average copy number of the reference genes in each sample by the average copy number of the reference genes of the control sample, and then dividing the number of transcripts measured (of each gene of interest) by this number, known as the normalisation factor (Vandesompele et al., 2002). Differences in copy number of cytokine genes between control and disease (OA) samples cohort were compared using the Whitney– Mann U-test. When detected, differences in cytokine expression (between the number of samples in which cytokine expression could be measured) were compared using Fisher’s Exact test. Statistical significance was determined as p < 0.05. Statistical tests were performed using statistical software (Minitab V14.1, Minitab Ltd., Coventry, UK) or a web-based statistical calculator (http://home.clara.net/sisa/fisher.htm).

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Fig. 1. Gene expression of interleukin-1b in canine articular tissues.

Fig. 2. Gene expression of interleukin-6 in canine articular tissues.

also detected in most samples of ruptured cruciate ligament (4 out of 5), but no expression was detected in control CCL ( p = 0.0476). A trend towards increased level of expression of IL-1b was detected in OA fat pad ( p = 0.09) when compared to control fat pad.

3. Results Gene expression (normalised transcript numbers per 200 mg of total RNA) in each tissue are presented for IL-1 (Fig. 1), IL-6 (Fig. 2), IL-8 (Fig. 3), IL-10 (Fig. 4) and IL-17 (Fig. 5). The results of the statistical tests are presented in Table 2. 3.1. IL-1b expression in OA tissues OA synovial membrane demonstrated both an increase in quantity of IL-1b expression ( p = 0.0038) and an increase in the number of samples in which expression was detected ( p = 0.0069) when compared to control synovial membrane. IL-1b expression was

Fig. 3. Gene expression of interleukin-8 in canine articular tissues.

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Fig. 4. Gene expression of interleukin-10 in canine articular tissues.

3.2. IL-6 expression in OA tissue IL-6 expression was significantly increased in OA synovial membrane ( p = 0.0043) when compared to control synovial membrane. IL-6 expression was

Fig. 5. Gene expression of interleukin-17 in canine articular tissues.

detected in all synovial membrane samples (OA, n = 8 and control, n = 5). Ruptured (OA) cruciate ligament also had a significant increase in the level of IL-6 gene expression when compared to control cruciate ligament ( p = 0.0112).

Table 2 Fisher’s Exact test P values (upper) and Mann–Whitney U-test P values (lower) Tissues

Canine cruciate ligament (control n = 5, ruptured (OA) n = 5)

Infrapatellar fat pad (control n = 5, OA n = 6)

Articular cartilage (control n = 5, OA n = 5)

Synovium (control n = 5, OA n = 8)

IL-1b FE MWU Number of samples Expressing IL-1b

0.047619 NA Control n = 0 OA n = 4

0.242424 0.0926 Control n = 2 OA n = 5

1 NA Control n = 0 OA n = 1

0.006993 0.0038 Control n = 1 OA n = 8

IL-6 FE MWU Number of samples Expressing IL-6

0.166667 0.0112 Control n = 2 OA n = 5

0.454545 0.1709 Control n = 4 OA n = 6

0.444444 0.2903 Control n = 3 OA n = 5

1 0.0043 Control n = 5 OA n = 8

IL-8 FE MWU Number of samples Expressing IL-8

1 0.2963 Control n = 5 OA n = 5

1 0.5228 Control n = 5 OA n = 6

1 0.2903 Control n = 3 OA n = 4

1 0.0338 Control n = 5 OA n = 8

IL-10 FE MWU Number of samples Expressing IL-10

0.206349 0.0746 Control n = 1 OA n = 1

1 0.3602 Control n = 4 OA n = 5

1 NA Control n = 0 OA n = 0

0.031857 0.0598 Control n = 1 OA n = 7

IL-17 FE MWU Number of samples Expression IL-17

0.166667 NA Control n = 0 OA n = 3

1 0.7739 Control n = 2 OA n = 3

1 NA Control n = 0 OA n = 1

0.51049 0.162 Control n = 3 OA n = 7

Cytokines

Significant values (P < 0.05) are presented in bold.

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3.3. IL-8 expression in OA tissue Control synovial membrane demonstrated a higher level of IL-8 expression when compared to OA synovial membrane ( p = 0.0338). All tissue samples expressed IL-8, which was the most abundantly expressed cytokine of the five cytokines evaluated in this study. 3.4. IL-10 expression in OA tissue IL-10 gene expression was detected in a significantly greater number of OA synovial membrane samples (n = 7) when compared to control synovial membrane (n = 1, p = 0.0318). Most of the OA samples showed expression of IL-10 (7 out of 8) and only 1 out of 5 control synovial membrane showed expression of IL10. There was also a trend towards expression being detected in an increased number of OA fat pad samples (4 out of 5) when compared to control (1 out of 5), although this was not significant ( p = 0.075). 3.5. IL-17 expression in OA tissue There was no significant difference in IL-17 expression across all of the articular tissue samples. IL-17 was the least abundantly expressed cytokine. 4. Discussion The primary pathways responsible for the imbalance between cartilage degradation and repair in OA are believed to be mediated through cytokines and chemokines (Fernandes et al., 2002), and blocking their action can abrogate their effects both in vitro and in vivo (Pelletier et al., 1997). The relative importance of different cytokines in the pathogenesis and development of OA, and the relative contributions of different articular tissues to the production of cytokines are presently not well defined. This study characterised the levels of expression of five cytokines in four different articular tissues from control and OA joints in dogs, through the absolute quantification of transcript numbers using RT-qPCR. IL-1b may be a primary mediator of cartilage catabolism in vitro, but expression of this gene appears to be primarily outside of cartilage itself (synovial membrane and cruciate ligament), which is consistent with the first report documenting cytokine production (protein) from articular tissues (Dingle, 1981). A similar pattern of expression is recognised for IL-6 which may reflect the induction of IL-6 expression by IL-1b, as has been reported in the synovial membrane

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(Bondeson et al., 2006) and periodontal ligament fibroblasts (Patil et al., 2006) in vitro. Contrary to what one may have expected on the basis of the known pro-inflammatory actions of IL-8, an increased expression of IL-8 was identified in the control synovial membrane when compared to OA synovial membrane. Previous reports have documented increased IL-8 expression in human OA synovial membrane when compared to normal synovial membrane (Furuzawa-Carballeda and Alcocer-Varela, 1999). The reasons for this finding are not clear. IL-8 was the most abundantly expressed cytokine across all tissue samples. Conversely, synovial fluid cell expression of IL-8 was found to be absent in normal dogs, and only present in 62% of OA samples, in a study of canine OA using a different assay (de Bruin et al., 2005), but reports evaluating human tissue indicate basal IL-8 mRNA expression in the cells and tissue types examined in this study, namely adipocytes (Sopasakis et al., 2005), chondrocytes (Borzi et al., 1999) and synovial tissue (Kraan et al., 2001). We could not substantiate a direct role for IL-17 in OA through differential expression, as IL-17 expression was either absent or at only extremely low levels in all control and OA tissues. Synovial membrane was the tissue in which the most abundant cytokine expression was detected in OA, with differential expression of IL-1b, IL-6 and IL-10. Cytokine expression was also identified in fat pad, which is not commonly evaluated in OA (Ushiyama et al., 2003), and cytokines were differentially expressed in ruptured cranial cruciate ligament. Our results indicate that cytokine expression in the OA joints is not restricted solely to synovial membrane or cartilage, thus the study of interactions between the multiple tissues which comprise the joint is important to understand the nature of disease. Synovial membrane contains a heterogeneous cell population, composed primarily of monocytes and fibroblasts, and the inflammatory cytokine production of such cells is consistent with the inflammatory response seen both histologically in OA synovial membrane (Fernandes et al., 2002) and at a molecular level in synovial cells in vitro (Bondeson et al., 2006). Histological studies demonstrate that the cranial cruciate ligament is covered with synovial membrane (Kobayashi et al., 2006). Thus, the increase in IL-1b and IL-6 gene expression noted in ruptured (OA) cruciate ligament may merely be a reflection of the mixture of cell types present within the tissue samples. The difficulty of separating different cell types in tissue samples for the accurate measurement of gene

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expression is well described (Day, 2006). Without recourse to histology based dissection techniques, this conundrum is difficult to resolve. However if this were true, we would have expected to observe the basal expression of IL-6 recognised in all the control cruciate ligament samples, as it was for control synovial samples. Furthermore, we have identified up-regulation of IL-6 expression in canine cruciate ligament fibroblasts in response to oncostatin M and TNF-a stimulation (unpublished findings), which supports the assertion that the up-regulation of IL-6 may have been at least in part due to the ligament component of the tissue sample rather than the synovial membrane covering. Whilst tissues from different origins were compared (cartilage, from the hip; fat pad, synovial membrane and ligament, from the stifle), this is a reflection of the surgical procedures used to procure the tissues. All tissues were removed as part of routine surgical treatment of clinical cases, and thus osteoarthritic articular cartilage is only removed from canine OA joints as part of an arthroplasty procedure (such as total hip replacement). Consequently, matching samples could not be obtained from the same joint for all the diseased samples. We have previously reported that changes in matrix associated gene expression in endstage OA cartilage is comparable between different joints in the same species and between species (Clements et al., 2006a). Ideally all tissue samples would have been obtained from the same joint, and an increased number of samples would have been assessed to verify or refute the findings of this study with greater statistical power. The variation observed in the data sets may also be attributed to the fact that the clinical samples from the stifle joints could have been obtained from patients at different stages of the same disease (cranial cruciate ligament rupture) or from patients with different pathologies leading to cruciate ligament rupture and secondary osteoarthritis. Additionally, further phenotyping of synovial inflammation (Lipowitz et al., 1985) or cartilage degradation (van der Sluijs et al., 1992) by histological measures, and grading of the clinical severity of disease may have reduced the heterogeneity of gene expression. Furthermore, studies of gene expression do not reflect the functional importance of each gene to the pathogenesis of a disease, although our results are consistent with previous studies of proteins measured in naturally occurring canine OA joint (Fujita et al., 2005). In conclusion, our findings suggest that changes in cytokine expression in OA joints are most commonly

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