HIF-1-Dependent IL-6 Activation in Articular Chondrocytes Initiating Synovitis in Femoral Head Ischemic Osteonecrosis

Yamaguchi, Ryosuke MD, PhD; Kamiya, Nobuhiro MD, PhD; Adapala, Naga Suresh PhD; Drissi, Hicham PhD; Kim, Harry K.W. MD, MS

Journal of Bone & Joint Surgery - American Volume:
doi: 10.2106/JBJS.15.01209
Scientific Articles
Abstract

Background: Ischemic osteonecrosis of the femoral head in children is associated with chronic hip synovitis and increased levels of the pro-inflammatory cytokine interleukin-6 (IL-6) in the synovial fluid due to unknown mechanisms. The purpose of this study was to investigate hypoxia-inducible factor-1 (HIF-1) activation as a molecular mechanism linking the induction of ischemic osteonecrosis to IL-6 production and the initiation of hip synovitis.

Methods: Ischemic osteonecrosis was surgically induced in the right femoral head of 6 piglets. A histologic score, synovial fluid volume, and IL-6 level were used to assess hip synovitis. IL-6 immunostaining of articular cartilage and synovial tissue was performed as well. To study the role of HIF-1 in IL-6 activation, in vitro experiments using an HIF-1α activator (deferoxamine) and inhibitor (HIF-1 small interfering-RNA [siRNA]) were carried out. Synovial cell responses to hypoxic chondrocyte-conditioned media with and without an IL-6 receptor blocker (tocilizumab) were assessed on the basis of IL-1β and tumor necrosis factor-alpha (TNF-α) gene expressions and with a synovial cell-proliferation assay.

Results: Induction of ischemic osteonecrosis produced hip synovitis and increased IL-6 levels in the synovial fluid. Immunostaining and protein analysis demonstrated articular chondrocytes as a source of increased IL-6 production. When articular chondrocytes were cultured under hypoxic conditions, significantly increased HIF-1α and IL-6 expressions were observed. Under hypoxic culture conditions, IL-6 gene expression was significantly increased by HIF-1α activation using deferoxamine and inhibited by HIF-1α inhibition using HIF-1 siRNA. Synovial cells exposed to hypoxic chondrocyte-conditioned medium showed significant increases in IL-1β and TNF-α gene expressions and cell proliferation, which were inhibited by the IL-6 receptor blocker tocilizumab.

Conclusions: Induction of ischemic osteonecrosis results in IL-6 production in the articular cartilage through an HIF-1-dependent pathway. IL-6 produced by hypoxic articular chondrocytes stimulates inflammatory cytokine responses in synovial cells, which were significantly decreased by tocilizumab.

Clinical Relevance: This study provides new insight into the inherent relationship between the induction of ischemia and the initiation of hip synovitis following ischemic osteonecrosis and suggests a potential therapeutic target in the treatment of the synovitis.

Author Information

1Center for Excellence in Hip Disorders, Texas Scottish Rite Hospital for Children, Dallas, Texas

2Department of Orthopaedic Surgery, University of Texas Southwestern Medical Center, Dallas, Texas

3Faculty of Budo and Sport Studies, Tenri University, Tenri, Nara, Japan

4Department of Orthopaedic Surgery, University of Connecticut Health Center, Farmington, Connecticut

E-mail address for H.K.W. Kim: Harry.Kim@tsrh.org

Article Outline

Legg-Calvé-Perthes disease is a childhood hip disorder associated with ischemic osteonecrosis of the femoral head. It affects 1 in 740 boys and 1 in 3,500 girls between the ages of 2 and 14 years1,2. The hip disorder can produce a permanent deformity of the femoral head and early-onset osteoarthritis. Chronic synovitis of the affected hip joint is a common feature of Legg-Calvé-Perthes disease and is associated with a poor clinical outcome3,4. The current paradigm is that the synovitis is due to joint irritation; hence, the strategy for its treatment relies mainly on nonspecific methods of symptom management such as decreasing activities and prescribing nonsteroidal anti-inflammatory drugs5. The fact that the mechanism responsible for hip synovitis following ischemic osteonecrosis of the femoral head has not been elucidated hinders the development of specific and effective treatments of the condition in patients with Legg-Calvé-Perthes disease or other osteonecrotic conditions.

Recent studies using serial gadolinium-enhanced magnetic resonance imaging (MRI) of patients with Legg-Calvé-Perthes disease revealed synovial effusion and synovial membrane enhancement of 1 to 2 years’ duration, demonstrating the chronic nature of the synovitis6,7. Importantly, analysis of 27 inflammatory cytokines in the synovial fluid of patients with Legg-Calvé-Perthes disease demonstrated a significant elevation of the pro-inflammatory cytokine interleukin-6 (IL-6)6. The other major pro-inflammatory cytokines were not elevated. Recent genetic association studies also suggest possible involvement of IL-6 polymorphism in Legg-Calvé-Perthes disease and juvenile chronic arthritis8,9.

Although substantial elevation of IL-6 has been observed in the synovial fluid of patients with Legg-Calvé-Perthes disease, the mechanism responsible for its elevation remains unknown. We conducted the present study to investigate the relationship between the induction of femoral head ischemia and IL-6 activation in a well-established piglet model of ischemic osteonecrosis of the femoral head and with in vitro experiments under hypoxic culture conditions. The piglet model produces a femoral head deformity and histopathologic changes resembling Legg-Calvé-Perthes disease5,10-15. It has been previously shown that a total disruption of blood supply to the femoral head induces severe hypoxia and cell necrosis in the deep layer of the articular cartilage16,17. However, the chondrocytes located in the superficial layer of the articular cartilage are subjected to a milder hypoxic stress and remain viable. Furthermore, these chondrocytes have been shown to respond to the hypoxic stress by activation of a master regulator of hypoxic cellular response, the hypoxia-inducible factor-1-alpha (HIF-1α) pathway, and its target genes11,13,16. Previous studies have shown HIF-1α activation to increase IL-6 production in a variety of cell types18-20. However, the role of HIF-1α activation in IL-6 production and the development of synovitis in ischemic osteonecrosis has not been studied, to our knowledge.

The purpose of this study was to investigate HIF-1 activation in articular chondrocytes as a molecular mechanism linking the induction of ischemic osteonecrosis to IL-6 production and the initiation of hip joint synovitis.

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Materials and Methods

The animal study was approved by the local institutional animal care and use committee. The use of discarded human articular cartilage samples was approved by the local institutional review board (Study ID: STU 171 012011-114).

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Induction of Ischemic Osteonecrosis of the Femoral Head

The piglet model of ischemic osteonecrosis has been described previously10-13. Twelve commercial Yorkshire piglets (6 to 8 weeks old) were used. Ischemic osteonecrosis of the whole femoral head was induced on the right side by applying suture ligatures tightly around the femoral neck (n = 6) to disrupt the femoral neck blood vessels and by resecting the ligamentum teres. In the sham-operation group (n = 6), the right femoral neck was exposed but no ligature was placed and the ligamentum teres was left intact (i.e., ischemia was not induced). No surgery was performed on the animals’ left femoral head, and these femoral heads were used as normal controls. At 2 and 4 weeks after the surgery (n = 3 per time point), synovial fluid was aspirated and synovial tissue and femoral heads were harvested for analysis.

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Histologic, Immunohistochemical, and Histomorphometric Analyses

Synovial tissue and femoral heads were harvested and fixed with 10% neutral buffered formalin. The femoral heads were decalcified with 10% EDTA (ethylenediaminetetraacetic acid). Both tissues were processed for paraffin embedding and microtome sectioning. An established histopathologic grading system21 was used by 3 independent observers to grade the severity of the synovitis in hematoxylin and eosin-stained sections. The number of chondrocytes and percentage of cloning cells present in the superficial and middle zones of the femoral head cartilage were quantified at 60× magnification under a Nikon Eclipse E800M microscope (Nikon) by observers who were blinded to the treatment group. Five randomly selected areas were counted in each zone. Immunohistochemical analysis was performed using a rabbit polyclonal primary antibody against IL-6, HIF-1α, or an isotype control IgG (immunoglobulin G) (Abcam). Horseradish peroxidase-conjugated secondary antibody (Millipore) and the 3,3′-diaminobenzidine tetrahydrochloride staining method were used.

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IL-6 Enzyme-Linked Immunosorbent Assay (ELISA)

An ELISA kit for porcine IL-6 (R&D Systems) was used to determine the IL-6 protein levels in the pig hip synovial fluid samples, the protein samples isolated from pig articular cartilage, and conditioned medium obtained from pig chondrocyte cultures.

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RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

Total RNA was isolated using an RNA isolation kit (Qiagen). Reverse transcription was performed using the SuperScript Preamplification System (Invitrogen). qRT-PCRs were performed using an ABI PRISM 7500 system and the TaqMan gene expression assay according to the manufacturer’s protocol (Applied Biosystems). Data analyses were performed by the Ct (ΔΔCt) method22. Applied Biosystems predesigned primers for human (IL-6, HIF-1α, and heat shock protein-90 [HSP90]) and pig (IL-1β, tumor necrosis factor-alpha [TNF-α], IL-6, HIF-1α, and HSP90) genes were used. The target quantity was normalized to the HSP90 mRNA level, which had more stable expression under hypoxic conditions than GAPDH (glyceraldehyde 3-phosphate dehydrogenase) and β-actin23-25.

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Isolation of Primary Chondrocytes and Synovial Cells

Pig articular chondrocytes were isolated from normal femoral cartilage of 3 piglets and used for chondrocyte-conditioned media experiments. In addition, primary human articular chondrocytes were isolated from the discarded human cartilage of 3 skeletally immature patients at the time of surgery25 and used for HIF-1α activation and inhibition experiments because a small interfering RNA (siRNA) for HIF-1 was commercially available for human cells and not for pig cells. The cartilage samples were digested overnight with collagenase type I (1 mg/mL) and dispase II (2 mg/mL) in Dulbecco modified Eagle medium (DMEM). The primary cells were cultured in minimum essential medium-alpha (MEMα; Invitrogen), 10% fetal bovine serum (FBS; Sigma-Aldrich), and antibiotics (100 IU/mL each of penicillin and streptomycin; Invitrogen) and incubated at 37°C, 5% CO2.

For the isolation of primary pig synovial cells, the synovial tissue of normal knee joints was dissected and was digested overnight with collagenase type I and dispase II in DMEM. Synovial cells were cultured in DMEM, 10% FBS, and antibiotics, and treated with recombinant IL-6 for 6 hours.

The primary cells from passages 2, 3, and 4 were used for all of the experiments under normoxic (21% oxygen) and hypoxic (1% oxygen) conditions maintained using a humidified Hypoxia Workstation (Coy Labs).

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HIF-1α Activation and Inhibition Experiments

For the HIF-1α activation study, deferoxamine (Hospira) was used to treat human articular chondrocytes for 24 hours. For the HIF-1α inhibition study, the siRNA method was used. Human articular chondrocytes grown to 50% to 60% confluence were transfected with the HIF-1 siRNA (100 nM) or the siControl scrambled RNA (Thermo Scientific) using the Lipofectamine RNAiMAX transfection reagent in Opti-MEM (Invitrogen). After 6 hours, the culture medium was recovered to a normal medium for an additional 18 hours. The transfected cells were then cultured under normoxic or hypoxic conditions for 6 hours.

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Normoxic and Hypoxic Chondrocyte-Conditioned Medium Studies

The effects of soluble factors produced by articular chondrocytes on the synovial cell inflammatory responses were investigated in 2 steps. For the first step—the preparation of the conditioned medium—pig articular chondrocytes were cultured in normoxic or hypoxic conditions for 72 hours followed by the collection of culture supernatant, which was filtered (0.20-μm pore) and was stored at −80°C. For the second step—exposure of the synovial cells to soluble factors in the articular chondrocyte-conditioned medium—pig primary synovial cells grown to 70% to 80% confluence were cultured in normoxic chondrocyte-conditioned medium or hypoxic chondrocyte-conditioned medium for 24 hours under normoxia. The IL-6 receptor blocker tocilizumab (100 μg/mL; Genentech) was used to block the soluble and membrane-bound IL-6 receptors in the culture medium and on the cell membrane. Inflammatory gene expressions and proliferation of synovial cells in response to treatments were determined at 24 or 48 hours (MTS assay; Promega). All in vitro experiments were performed in triplicate.

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Statistical Analysis

Statistical analyses were performed using the Student t test for comparisons of 2 groups and using analysis of variance (ANOVA) and the Tukey post hoc test for comparison of ≥3 groups. A p value of <0.05 was considered significant.

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Results

Hip Synovitis and Synovial Fluid IL-6 Elevation Following Induction of Ischemic Osteonecrosis

The induction of osteonecrosis resulted in hip synovitis as evidenced by significantly higher histologic synovitis scores in the osteonecrosis group compared with those in the normal cartilage (all p < 0.001, Figs. 1-A and 1-B). The volume of synovial fluid collected from the hip joint in the osteonecrosis group at the time of sacrifice was significantly greater than that in the normal group (mean and standard deviation, 2.9 ± 0.5 mL versus 0.3 ± 0.04 mL, p = 0.0002; Fig. 1-C). Analysis of the synovial fluid showed significantly elevated IL-6 protein levels in the osteonecrosis group compared with the normal group (p = 0.02, Fig. 1-D).

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IL-6 Expression in Articular Chondrocytes and Synovial Cells

Articular chondrocytes in the osteonecrosis group showed a greater number of IL-6-positive cells compared with the sham-operation or normal group (Fig. 2-A). The osteonecrosis group demonstrated a significantly greater number of chondrocytes and higher percentage of cloning cells than the sham-operation and normal groups (all p < 0.01, Figs. 2-B and 2-C). The increased levels of IL-6 protein in the articular cartilage of the osteonecrosis group were confirmed by ELISA (p = 0.01, Fig. 2-D). The synovium in the osteonecrosis group also showed a greater number of IL-6-positive cells compared with the sham-operation or normal group (Fig. 3-A).

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HIF-1α Expression in Articular Chondrocytes and Synovial Cells

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.

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HIF-1α-Mediated IL-6 Activation in Articular Chondrocytes

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.

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IL-6 Induction of Inflammatory Cytokine Response and Proliferation of Synovial Cells

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).

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Hypoxic Chondrocyte-Conditioned Medium Stimulating Synovial Inflammatory Cytokine Response

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.

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Discussion

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

Disclosure: This study was supported by an intramural grant from the Texas Scottish Rite Hospital for Children. On the Disclosure of Potential Conflicts of Interest forms, which are provided with the online version of the article, one or more of the authors checked “yes” to indicate that the author had other relationships or activities that could be perceived to influence, or have the potential to influence, what was written in this work.

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