Since HIF-1α has previously been shown to be activated in articular chondrocytes following the induction of ischemic osteonecrosis in the piglet model16,26, we examined HIF-1α immunostaining in articular chondrocytes and synovial cells. The osteonecrosis group demonstrated a greater number of HIF-1α-positive cells compared with the normal group (Fig. 4-A). In in vitro experiments, articular chondrocytes showed significant increases in HIF-1α and IL-6 mRNA expressions (p < 0.01 and 0.005) and in IL-6 protein level (p < 0.001) under hypoxic (1% oxygen) culture conditions compared with the values under normoxic (21% oxygen) culture conditions (Figs. 4-B, 4-C, and 4-D). However, hypoxic culture conditions did not increase HIF-1α and IL-6 expression in the synovial cells (p = 0.12 and 0.96, Figs. 3-B and 3-C). Taken together, these results suggest that, following the induction of ischemic osteonecrosis, hypoxic stress primarily induces IL-6 production in articular chondrocytes but not in synovial cells.
Treatment of human articular chondrocytes with the HIF-1α activator deferoxamine resulted in a dose-dependent increase in the IL-6 gene expression (all p < 0.01, Fig. 5-A). In the inhibition study, HIF-1 siRNA reduced HIF-1α gene expression in articular chondrocytes under normoxic and hypoxic conditions (p < 0.001 and 0.002, Fig. 5-B). Furthermore, HIF-1 siRNA decreased IL-6 gene expression under both normoxic and hypoxic conditions (p < 0.01 and 0.002, Fig. 5-C). These results indicate that HIF-1α mediates IL-6 production in articular chondrocytes under hypoxic stress.
When the pig synovial cells were treated with recombinant IL-6, the mRNA levels of the proinflammatory cytokines IL-1β and TNF-α were significantly increased compared with the levels in the untreated group (p = 0.01 and 0.02, Fig. 6-A). The proliferation of synovial cells was also increased at 24 and 48 hours after treatment with recombinant IL-6 (p = 0.01 and 0.03, Fig. 6-B).
We then investigated the hypothesis that IL-6 released from hypoxic chondrocytes following ischemia induction stimulates the inflammatory responses in synovial cells. Normoxic and hypoxic chondrocyte-conditioned media were used to treat the synovial cells. Hypoxic chondrocyte-conditioned medium significantly increased expressions of IL-1β and TNF-α compared with the expressions in the normoxic chondrocyte-conditioned medium (p = 0.02 and 0.04, Fig. 7-A). Treatment of synovial cells with hypoxic chondrocyte-conditioned medium also significantly increased the proliferation of synovial cells compared with that in the normoxic chondrocyte-conditioned medium (p = 0.02, Fig. 7-B).
To determine whether IL-6 in hypoxic chondrocyte-conditioned medium is responsible for these effects, an IL-6 receptor blocker (tocilizumab) was used to block the effects of IL-6 in the conditioned media. In the presence of tocilizumab, the expressions of IL-1β and TNF-α were significantly decreased (both p = 0.03, Fig. 7-A). Tocilizumab also significantly decreased the synovial cell proliferation observed in response to the treatment with hypoxic chondrocyte-conditioned medium (p = 0.03, Fig. 7-B). Taken together, these results indicate that IL-6 released from hypoxic articular chondrocytes stimulates the inflammatory response from synovial cells and this response can be abated by tocilizumab.
Hip synovitis is a common feature of Legg-Calvé-Perthes disease, causing pain, loss of hip motion, and increased susceptibility to femoral head deformity. A recent serial gadolinium-enhanced MRI study showed that hip synovitis in patients with Legg-Calvé-Perthes disease is chronic in nature, with some patients showing evidence of the synovitis after more than 2 years of follow-up6. The study also demonstrated a significant elevation of a pro-inflammatory cytokine, IL-6, in the synovial fluid of patients with Legg-Calvé-Perthes disease. The mechanisms responsible for the initiation and chronic persistence of synovitis following the induction of ischemic osteonecrosis are unknown. We performed the present study to investigate the relationship between the induction of ischemic osteonecrosis and the production of IL-6 in a well-established piglet model of Legg-Calvé-Perthes disease. The results of the study provide new evidence that hypoxic stress induces IL-6 production from articular chondrocytes through the activation of a master regulator of hypoxic cellular response, HIF-1α. We also showed that IL-6 derived from hypoxic chondrocytes can induce an inflammatory cytokine response in synovial cells, which can be abated by an IL-6 receptor blocker, tocilizumab.
We found an inherent link between the induction of femoral head ischemia and the production of IL-6 from articular chondrocytes due to HIF-1α activation, which stimulates an inflammatory response from synovial cells (Fig. 8). While other mechanisms may be involved with the initiation and propagation of hip synovitis following ischemic osteonecrosis, HIF-1α activation in articular chondrocytes may be one of the earliest mechanisms responsible for the IL-6 upregulation. Indeed, a previous study using a hypoxyprobe staining method showed a rapid induction of a severe hypoxic state (pO2 < 10 mm Hg) in the deeper layer of the articular cartilage within 4 hours after the induction of ischemic osteonecrosis in the piglet model15,16. The chondrocytes located superficial to the deep layer of the cartilage were found to respond to the hypoxic stress by HIF-1α activation, which in turn upregulates IL-6 expression from the chondrocytes.
The results of our in vitro studies further support the hypothesis that hypoxic articular chondrocytes rather than synovial cells are the initial source of IL-6 in the synovial fluid following the induction of ischemic osteonecrosis, because hypoxic culture conditions induced both HIF-1α and IL-6 expressions in articular chondrocytes but not in synovial cells. IL-6 released by the hypoxic chondrocytes significantly increased the expression of the pro-inflammatory cytokines IL-1β and TNF-α and increased proliferation of synovial cells. Given that the synovial fluid bathes the articular cartilage and the synovium, we propose that the hypoxic chondrocytes produce IL-6, which contributes to the elevation of IL-6 in the synovial fluid. Its elevation subsequently induces synovitis through IL-1β and TNFα activation in synovial cells and synovial cell proliferation.
It is noteworthy that an IL-6 receptor blocker, tocilizumab, reduced the inflammatory cytokine response and cell proliferation in synovial cells exposed to the hypoxic chondrocyte-conditioned medium. These results warrant future in vivo studies to investigate the therapeutic potential of IL-6 blockade for decreasing synovitis following ischemic osteonecrosis. Currently, there are no specific treatments for hip synovitis associated with Legg-Calvé-Perthes disease or any other forms of ischemic osteonecrosis. Several multicenter randomized clinical trials have demonstrated acceptable safety and significant efficacy of tocilizumab for chronic inflammatory joint disorders such as rheumatoid arthritis in the adult population and systemic idiopathic juvenile arthritis and polyarticular idiopathic juvenile arthritis in the pediatric population27-31. In addition to decreasing synovitis, IL-6 receptor blockade may have positive effects in terms of improving the remodeling of the necrotic bone. Previous studies using the piglet model of ischemic osteonecrosis have shown a predominance of bone resorption and a lack of new bone formation during the remodeling of the necrotic bone10,32,33. Since IL-6 is known to increase the resorptive activity of osteoclasts34-36 and inhibit the differentiation of osteoblasts36,37, future investigation of the therapeutic role of IL-6 receptor blockade in synovitis and bone remodeling following ischemic osteonecrosis is warranted.
In summary, induction of ischemic osteonecrosis results in IL-6 activation in the articular cartilage through the HIF-1 signaling pathway. IL-6 produced by hypoxic articular chondrocytes stimulates synovial cell proliferation and an inflammatory cytokine response, which were significantly decreased by IL-6 receptor blockade.
NOTE: The authors thank Amanda McLerran for animal care and surgical assistance, Reuel Cornelia and Richard Banlaygas for histologic preparation, and Ila Oxendine and Yang Li for experimental assays.
Investigation performed at the Texas Scottish Rite Hospital for Children, Dallas, Texas
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