Elevation of Proinflammatory Cytokine HMGB1 in the Synovial Fluid of Patients With Legg‐Calvé‐Perthes Disease and Correlation With IL ‐6

Figures

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  Fig 1. Increase of high‐mobility group box 1 (HMGB1) protein levels in synovial fluid of patients with Legg‐Calvé‐Perthes disease (LCPD). (A) HMGB1 protein levels were much higher in the LCPD group compared with the control group (control: #1–3; LCPD: #4–8). (B) The integrated density values of each sample from Western blot were measured. The density in the LCPD group was significantly higher than that in the control group. **p < 0.01 (control: n = 3; LCPD: n = 5; the density of #1 was set as 1.0).

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  Fig 2. Increased high‐mobility group box 1 (HMGB1) protein in synovial fluid of patients with Legg‐Calvé‐Perthes disease (LCPD). HMGB1 protein levels (ng/mL) in synovial fluid were measured. The mean HMGB1 value was threefold higher in patients with LCPD compared with that in control patients. *p < 0.05 (control: n = 5; LCPD: n = 8).

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  Fig 3. Positive correlation between high‐mobility group box 1 (HMGB1) and IL‐6 protein levels in synovial fluid of patients with Legg‐Calvé‐Perthes disease (LCPD). IL‐6 protein levels (pg/mL) were measured using synovial fluid shown in Fig. 1. The correlation of IL‐6 protein values and the HMGB1 relative density values is shown. Each scatter plot shows one human sample. There is a high and statistically significant correlation between the values of IL‐6 and HMGB1 proteins (Pearson correlation coefficient 0.94, p < 0.001, R 2 = 0.87; n = 8).

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  Fig 4. Increased high‐mobility group box 1 (HMGB1) protein in articular cartilage following ischemic osteonecrosis (ON) induction in a mouse model of juvenile ischemic ON. (A) HMGB1 protein was immunohistochemically analyzed with anti‐HMGB1 antibody. HMGB1 immuno‐staining intensity was much higher in the articular cartilage after ischemic induction at day 7 in the ON‐right group compared with the sham‐right group. The HMGB‐1 immuno‐staining was not detectable in the contralateral ON‐left and sham‐left groups (ON: n = 4; sham: n = 4). R = Right side; L = left side. Bars: 50 μm. (B) HMGB1 protein level was analyzed by Western blot. After 24 hours of ON or sham surgery, the articular cartilage of the distal femur was harvested, and protein was extracted. HMGB1 protein was highly expressed in the ON‐right group compared with the sham‐right group (ON: n = 4; sham: n = 4). Note that HMGB1 protein was faintly expressed in the contralateral sides (ie, sham‐left, ON‐left). R = Right side; L = left side.

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  Fig 5. Chondrocytes exposed to hypoxic and oxidative stresses produced high‐mobility group box 1 (HMGB1) and IL‐6 proteins in culture supernatant. (A) Human primary chondrocytes were cultured in normoxia or hypoxia for 24 hours, and culture supernatant was collected from each condition. The concentration of HMGB1 protein was significantly higher in the supernatant of hypoxic than that of normoxic condition. n = 3 per each group, *p < 0.05. (B) Hydrogen peroxide (H2O2; 0 to 200μM) was added to the culture of primary chondrocytes for 24 hours. The concentration of HMGB1 protein in the culture supernatants was increased in a dose‐dependent manner. (C) Human primary chondrocytes were cultured in normoxia or hypoxia for 24 hours, and culture supernatants were collected from each condition. The concentration of IL‐6 protein was significantly higher in the supernatant of hypoxic than that of normoxic condition. n = 3 per each group, *p < 0.05. (D) H2O2 (0 to 200μM) was added to the culture of primary chondrocytes for 24 hours. The concentration of IL‐6 protein in the culture supernatants was increased in a dose‐dependent manner.

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  Fig 6. Overexpression of high‐mobility group box 1 (HMGB1) protein in the culture supernatant using chondrocytes. (A) ATDC5 cells were transfected with HMGB1 or mock plasmid and cultured for 48 hours. Cells were harvested and protein was extracted. HMGB1 protein was detectable in the HMGB1‐transfected cells; it was undetectable in the mock‐transfected cells. Representative image is shown (n = 3 per each group). (B) HMGB1 protein was detectable in the culture supernatants from HMGB1‐transfected ATDC5 cells; it was undetectable in the mock‐transfected culture supernatants, as derived from the culture represented by Fig. 6A. Representative image is shown (n = 3 per each group). (C) Human chondrocytes were transfected with HMGB1 or mock plasmid and cultured for 48 hours. Expression levels of IL‐6 in the HMGB1‐transfected cells were significantly higher than that in the mock‐transfected cells, as derived from the culture represented in Fig. 6A. *p < 0.05 (mock: n = 4; HMGB1: n = 4). (D) Human chondrocytes were transfected with HMGB1 or mock plasmid and cultured for 48 hours, and culture supernatants were collected from each condition. The concentration of IL‐6 protein was significantly…

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  Fig 7. Inhibition of high‐mobility group box 1 (HMGB1) by short interfering RNA (siRNA) in human chondrocytes. (A) HMGB1 expression was significantly reduced by 90% in the siHMGB1‐transfected cells compared with the siControl‐transfected cells. The value from the nontreatment cells (ie, None) was set as 1.0. **p < 0.01. (B) IL‐6 expression was unchanged between the siHMGB1‐transfected and siControl‐transfected cells. (C) IL‐1β expression was significantly reduced by 91% in the siHMGB1‐trenasfected cells compared with the siControl‐transfected cells. *p < 0.05. (D) The concentration of IL‐6 protein in the supernatants was unchanged between the siHMGB1‐transfected and siControl‐transfected cells (n = 3 per each group in the Fig. 7).

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  Fig 8. Positive correlations among IL‐6, IL‐1β, and TNF‐α protein levels in the synovial fluid of patients with Legg‐Calvé‐Perthes disease (LCPD). (A–C) Protein levels of IL‐6, IL‐1β, and TNF‐α (pg/mL) in synovial fluid of patients with LCPD (n = 13) were measured by a multicytokine assay kit. Each scatter plot shows one human sample. Significant correlations were observed between IL‐6 and IL‐1β (Pearson correlation coefficient 0.74, p = 0.004, R 2 = 0.55), between IL‐6 and TNF‐α (Pearson correlation coefficient 0.80, p = 0.001, R 2 = 0.64), and between IL‐1β and TNF‐α (Pearson correlation coefficient 0.99, p < 0.0001, R 2 = 0.98). (D) A possible model of disease mechanism following ischemic osteonecrosis. Proinflammatory cytokines high‐mobility group box 1 (HMGB1) and IL‐6 were produced by articular chondrocytes following ischemic induction and were amplified via synovium, resulting in the increase of cytokines in the synovial fluid including IL‐1β and TNF‐α.