STIMULATION OF CYTOKINE AND CHEMOKINE EXPRESSION BY HUMAN ARTICULAR CHONDROCYTES IN RESPONSE TO FIBRONECTIN FRAGMENTS

METHODS Human articular chondrocytes, isolated from normal ankle cartilage obtained from tissue donors, were treated with the 110kD FN-f (0.5-1µM) in serum-free monolayer culture as previously described (2). RNA was collected at 6 and 16 hours after stimulation and expression of various cytokine genes was analyzed by cDNA microarray using the Clontech human cytokine array (268 genes). Conditioned media was collected 24 hours after FN-f treatment and used for protein array with the RayBio human cytokine protein array (23 cytokines). It should be noted that this array contained IL-1α but not IL-1s, the latter of which had previously been shown to be upregulated by FN -f (2). Expression of the cytokines identified by the array studies was further examined by RT PCR in cells stimulated with FN-f and compared to cells stimulated with IL-1s (2ng/ml) and TNFα (10 ng/ml). GAPDH was used as a control and ratios of cytokine/GAPDH were calculated. RESULTS Compared to untreated control cultures, stimulation by FN -f resulted in up regulation (>2 -fold ratio FN-f/control) of 19/268 (7.1%) of the cytokine and chemokine genes at 6 hours and 22/268 (8.2%) at 16 hours. The gene array results were then compared to results using a cytokine protein array. The most highly expressed cytokines stimulated by FN-f and identified by both gene and protein arrays, were IL-6, IL -8, MCP-1, and GRO-s (Fig.1). Expression of the cytokines and chemokines identified on gene and protein array was further studied using RT -PCR. FN-f (0.5µM) treatment for 6 hrs. stimulated > 1.5 -fold expression of IL-6, IL -8, GRO-s, and MCP -1 when compared to unstimulated controls. Stimulation of IL-1s expression was also confirmed, while there was little increase in RNA levels for TNFα (which was on the protein array but not detect ed). In addition to GRO -s, 2 other GRO family members, GRO-α and GRO–γ, were identified by RT -PCR and were also increased by FN -f treatment. Stimulation of chondrocytes with IL-1s (2 ng/ml) resulted in a similar increase in RNA levels for IL-6, IL -8, MCP -1 and GROfamily members as 0. 5 µM FN-f. TNFα (10 ng/ml) stimulated expression of GRO-α and MCP -1 but not GRO-s or GRO-γ. CONCLUSION The ability of FN -f to stimulate chondrocyte expression of the pro-inflammatory cytokines IL-1, IL-6, IL-8, MCP-1 and GRO family members suggests that damage to the cartilage ECM resulting in fragment formation is capable of inducing a pro-inflammatory state that can be responsible for further progressive matrix destruction. The GRO family proteins are members of the IL-8 superfamily and along with MCP-1 are classified as chemokines due to their known chemotactic activity. IL-8 and GRO proteins are CXC chemokines which attract neutrophils while MCP-1 is a C -C chemokine which attracts macrophages. FN-f stimulated expression of chondrocyte chemokine expression may be particularly important in the pathogenesis of rheumatoid arthritis where pannus formation plays an important role in cartila ge destruction. IL-1, IL -6, IL -8, MCP -1, and GRO-α have also been demonstrated in OA cartilage and have catabolic and anti-anabolic effects. The results of this study suggest that targeting the signaling pathways activated by FN-f may be an effective means of inhibiting production of multiple mediators of cartilage destruction.