Abstract
BACKGROUND
Although osteoarthritis (OA) is not an inflammatory arthritis, a characteristic feature of OA is increased production of pro-inflammatory cytokines, such as interleukin 1beta (IL-1b), by articular chondrocytes. In fact, the degree of articular inflammation is often associated with disease progression; indicating that this process probably contributes to articular damage. Suppressor of cytokine signalling (SOCS) proteins are, as the name suggests, inhibitors of cytokine signalling that function via the JAK/STAT pathway (Janus kinase/signal transducers and activators of transcription). Eight SOCS proteins, SOCS1-SOCS7 and CIS-1 (cytokine-inducible SH2-domain-1 with similar structure to the other SOCS proteins) have been identified, of which, SOCS1-3 and CIS-1 are the best characterised. Reduced expression of SOCS proteins would be predicted to result in increased cytokine responsiveness and thereby could contribute to OA pathology.
OBJECTIVES
1) To compare the expression of SOCS1-3 and CIS-1 in normal and OA human articular chondrocytes and 2) to analyze the effects of IL-1b on SOCS1-3 and CIS-1 mRNA expression.
METHODS
Femoral heads were obtained after joint replacement surgery due to OA or following a fracture of the neck of femur (#NOF). The latter patients typically suffer from osteoporosis and their cartilage is widely used as a suitable non-OA control. Chondrocytes from the surface layer of OA or the deep zone of #NOF cartilage were isolated by sequential treatment with trypsin, hyaluronidase and collagenase B. Total RNA was extracted using the Qiagen AllPrep RNA mini kit. Primers were designed for SOCS1-3 and CIS-1 and relative gene expression was determined by real-time RT-PCR (Sybr-green method), normalized to GAPDH. Alternatively, non-OA chondrocytes were isolated from #NOF cartilage and cultured in DMEM/F12/5% FCS, with or without IL-1b plus oncostatin M (OSM, both 2.5 ng/ml) for 3-5 weeks until confluent. Total RNA was extracted as above and the effect of IL-1b on gene expression was determined by qPCR.
RESULTS
Expression of SOCS1 and SOCS3 was similar in OA and #NOF chondrocytes. However the expression of SOCS2 and CIS-1 was reduced 13-fold in OA samples compared to #NOF samples (n=5, each group). Expression of one OA patient was set to =1 to determine relative expression. In #NOF chondrocytes, relative expression of SOCS2 was 24.5±20.4 versus 2.5±1.9 in OA chondrocytes and for CIS-1 the values were 26.5±19.5 versus 2.5±1.8 (p<0.01). The in vitro experiments showed that treatment of control #NOF chondrocytes with IL-1b+OSM mimicked the situation in OA: Expression of SOCS2 in cytokine-treated cultures was only 15±10% of that in control cultures and CIS-1 expression was reduced to 16±13% (P<0.01).
CONCLUSIONS
To the authors' knowledge this is the first demonstration of reduced expression of SOCS2 and CIS-1, but not SOCS1 or SOCS3, in osteoarthritis. As the SOCS proteins are attenuators of cytokine-mediated processes, reduced expression would be expected to accelerate any inflammatory processes. The fact that IL-1b itself reduces the expression of its suppressors of signalling suggests a potentially damaging positive feedback mechanism that could play a role in OA pathology.
