SAT0443 In Vitro Effects of Canakinumab on Human Osteoarthritic Chondrocytes
Authors: S. Cheleschi, L. Cantarini, N.A. Pascarelli, O.M. Lucherini, G.M. Guidelli, G. Collodel, M. Galeazzi, A. Fioravanti
Journal: Annals of the Rheumatic Diseases (2014)

<h3>Background</h3> Canakinumab is a human IgGκ monoclonal antibody targeting Interleukin (IL)-1β; its action involves the neutralization of the signaling of IL-1β, the most important cytokine in the pathogenesis of Osteoartrhitis (OA). In response to IL-1β, chondrocytes secrete other proinflammatory cytokines, neutral metalloproteinases (MMPs), nitric oxide (NO); furthermore IL-1β inhibits chondrocytes proliferation and induces apoptosis. <h3>Objectives</h3> The aim of our study was to evaluate the possible <i>in vitro</i> effects of canakinumab on human OA chondrocytes cultures cultivated in the presence or absence of tumor necrosis factor (TNF)-α. <h3>Methods</h3> Chondrocytes, from femoral cartilage of five OA patients who underwent surgery for hip prostheses, were isolated using sequential enzymatic digestion. Primary cultures so obtained were incubated with canakinumab (1μg/ml and 10μg/ml) alone or with TNF-α (10ng/ml). We evaluated after 48h cell viability, release of proteoglycans (PG) and NO in culture medium, inducible nitric oxide synthase (iNOS) and MMP-1,3,13 expression, the percentage of apoptosis and necrosis. After 24h we performed IL-1β dosage (ELISA). Finally, we used a transmission electron microscope (TEM) for morphological assessment. <h3>Results</h3> Canakinumab alone at the two concentrations studied hadn9t cytotoxic effect and didn9t modify significantly IL-1β levels in the culture medium. TNF-α caused a significant decrease of the percentage of viable cell (P&lt;0.001) and a significant increase of IL-1β. Both concentrations of canakinumab significantly restored the cell viability and reduced IL-1β production in TNF-α stimulated chondrocytes. No significant modification of PG levels was observed in the cultures treated with canakinumab alone; TNF-α determined a significant decrease (P&lt;0.001) in PG levels restored by canakinumab in a significant dose-dependent manner. Incubation of chondrocytes with canakinumab alone didn9t affect nitrite release; TNF-α induced a significant increase (P&lt;0.001) in NO production that was significantly (P&lt;0.01) reduced after co-incubation with TNF-α and canakinumab at two concentrations used. The data of the NO were confirmed by the immunocytochemistry assay for iNOS. TNF-α stimulated chondrocytes displayed a significant increase of MMP-1,3,13 gene expression; a significant reduction was shown after co-treatment with TNF-α and canakinumab. Our experiments confirmed the pro-apoptotic effect of TNF-α and demonstrated a protective role of canakinumab at two concentrations examined. The biochemical results were further confirmed by TEM. <h3>Conclusions</h3> It is generally accepted that IL-1β and TNF-α are the pivotal cytokines involved in OA physiopathology. Hence, the neutralization of these cytokines appears to be a logical development for OA therapy. In the present study we showed, for the first time, that canakinumab counteracts the negative effect of TNF-α on OA chondrocyte cultures, probably inhibiting IL-1β signaling. <h3>References</h3> Dhimolea E. Mabs 2010;2:3-13. Fioravanti et al. J Pharmacol Sci 2012;20:6-14. <h3>Disclosure of Interest</h3> None declared <h3>DOI</h3> 10.1136/annrheumdis-2014-eular.5066