The impact of early intra-articular administration of interleukin-1 receptor antagonist on lubricin metabolism and cartilage degeneration in an anterior cruciate ligament transection model

Abstract

Objective: Study the impact of intra-articular interleukin-1 receptor antagonist (IL-1 ra) treatment on lubricin biosynthesis following anterior cruciate ligament transection (ACLT) in the rat and evaluate the effect of combined IL-1 ra and recombinant human lubricin (rhPRG4) treatments on chondrocyte apoptosis.

Methods: ACLT was performed in male Lewis rats. Treatments included IL-1 ra or vehicle (n = 36 in each group). IL-1 ra intra-articular dosing was performed on days 1, 3, 5 and 7 following ACLT using Anakinra (150 mg/ml; 40 μl). At 3 and 5 weeks, animals were sacrificed and RNA was isolated. Histological analyses included Safranin O and H&E. Lubricin synovial fluid (SF) lavage concentrations were determined at 5 weeks. ACLT animals were treated with a single injection of vehicle, IL-1 ra (75 mg/ml; 40 μl), rhPRG4 (200 μg/ml; 40 μl), or IL-1 ra + rhPRG4 (75 mg/ml + 200 μg/ml; 40 μl) (n = 6 in each group) on day 7 following ACLT and cartilage was probed for cleaved caspase-3 at 5 weeks.

Results: IL-1 ra treatment improved lubricin expression (P < 0.001) and lubricin SF lavage concentrations in the IL-1 ra group was higher (P = 0.005) than the vehicle. IL-1 ra treatment reduced cartilage and synovial scores (P < 0.001) compared to vehicle. IL-1 ra and rhPRG4 acted synergistically to reduce caspase-3 positive chondrocytes (P < 0.001) compared to individual treatments.

Conclusion: IL-1 ra treatment preserved lubricin following ACLT and a combined treatment of IL-1 ra + rhPRG4 may act synergistically to reduce cartilage catabolism.

Keywords: Interleukin-1 receptor antagonist; Lubricin; Posttraumatic osteoarthritis.

Figure 1

Quantitative lubricin gene expression in articular cartilage, lubricin cartilage immunostaining and synovial fluid (SF) lavage lubricin concentrations following anterior cruciate ligament transection (ACLT) and intra-articular treatment with PBS or interleukin-1 receptor antagonist (IL-1 ra) at 3 and 5 weeks following ACLT.

1. Relative lubricin cartilage expression in control and ACLT joints compared to contra-alteral joints and normalized to glyceraldehyde-3-phosphare dehydrogenase (GAPDH) and received intra-articular injections of PBS or IL-1 ra. Individual data points are presented and the median value is highlighted with an “X”.

   *Indicates that lubricin cartilage expression in control joints was significantly higher compared to 3-week and 5-week ACLT treated with PBS or IL-1 ra (p<0.001).

   **Indicates that lubricin cartilage expression in 3 week ACLT joints treated with IL-1 ra was significantly higher compared with 3 and 5 week ACLT joints treated with PBS (p=0.002), (p=0.001).

   ***Indicates that lubricin cartilage expression in 5 week ACLT joints treated with IL-1 ra was significantly higher compared with 5 week ACLT joints treated with PBS (p=0.001).

2. mAb 9G3 immunostaining for lubricin from (A) control, (B) 3-week ACLT joints treated with PBS, (C) 3-week ACLT joints treated with IL-1 ra, (D) 5-week ACLT joints treated with PBS, and (E) 5-week ACLT joints treated with IL-1 ra. Arrows point to intense lubricin staining on the surface of articular cartilage and in superficial zone chondrocytes compared to 3 and 5 week ACLT joints treated with PBS. Scale = 50 µm.

3. Urea-adjusted lubricin SF lavage concentrations in control, 5-week ACLT animals following treatment with PBS or IL-1 ra (n=6 in each group). Individual data points are presented and the median value is highlighted with an “X”.

   *Indicates that lubricin SF lavage concentrations in control animals were significantly higher compared with 5-week ACLT joints treated with PBS (p<0.001).

   **Indicates that lubricin SF lavage concentrations in 5-week ACLT animals treated with IL-1 ra was significantly higher compared with 5-week ACLT animals treated with PBS (p=0.005).

Figure 1

Quantitative lubricin gene expression in articular cartilage, lubricin cartilage immunostaining and synovial fluid (SF) lavage lubricin concentrations following anterior cruciate ligament transection (ACLT) and intra-articular treatment with PBS or interleukin-1 receptor antagonist (IL-1 ra) at 3 and 5 weeks following ACLT.

1. Relative lubricin cartilage expression in control and ACLT joints compared to contra-alteral joints and normalized to glyceraldehyde-3-phosphare dehydrogenase (GAPDH) and received intra-articular injections of PBS or IL-1 ra. Individual data points are presented and the median value is highlighted with an “X”.

   *Indicates that lubricin cartilage expression in control joints was significantly higher compared to 3-week and 5-week ACLT treated with PBS or IL-1 ra (p<0.001).

   **Indicates that lubricin cartilage expression in 3 week ACLT joints treated with IL-1 ra was significantly higher compared with 3 and 5 week ACLT joints treated with PBS (p=0.002), (p=0.001).

   ***Indicates that lubricin cartilage expression in 5 week ACLT joints treated with IL-1 ra was significantly higher compared with 5 week ACLT joints treated with PBS (p=0.001).

2. mAb 9G3 immunostaining for lubricin from (A) control, (B) 3-week ACLT joints treated with PBS, (C) 3-week ACLT joints treated with IL-1 ra, (D) 5-week ACLT joints treated with PBS, and (E) 5-week ACLT joints treated with IL-1 ra. Arrows point to intense lubricin staining on the surface of articular cartilage and in superficial zone chondrocytes compared to 3 and 5 week ACLT joints treated with PBS. Scale = 50 µm.

3. Urea-adjusted lubricin SF lavage concentrations in control, 5-week ACLT animals following treatment with PBS or IL-1 ra (n=6 in each group). Individual data points are presented and the median value is highlighted with an “X”.

   *Indicates that lubricin SF lavage concentrations in control animals were significantly higher compared with 5-week ACLT joints treated with PBS (p<0.001).

   **Indicates that lubricin SF lavage concentrations in 5-week ACLT animals treated with IL-1 ra was significantly higher compared with 5-week ACLT animals treated with PBS (p=0.005).

Figure 2

Impact of IL-1 ra treatment on cartilage and synovial histopathologies and urinary CTXII release following anterior cruciate ligament transection (ACLT) in the rat.

1. Representative Safranin-O (top panel) stained cartilage and Hematoxylin & Eosin (H&E; bottom panel) stained synovium from control animals and animals that underwent ACLT followed by intra-articular injection of PBS on days 1, 3, 5 and 7 (PBS) or IL-1 ra (IL-1 ra). The arrow points to synovial thickening and infiltration of inflammatory cells in the PBS-treated group. Scale = 50 µm.

2. Modified OARSI scores of control (n=5), PBS-treated ACLT animals (n=9) and IL-1 ra-treated ACLT animals (n=9). Individual data points are presented and the median value is highlighted with an “X”.

   *Indicates that OARSI scores of the PBS group was significantly higher than OARSI scores of control or the IL-1 ra groups (p<0.001).

3. Synovial histopathology scores of control (n=5), PBS-treated ACLT animals (n=9) and IL-1 ra-treated ACLT animals (n=9). Individual data points are presented and the median value is highlighted with an “X”.

   *Indicates that synovial histopathology scores of PBS-treated ACLT animals were significantly higher than control and IL-1 ra-treated ACLT animals (p<0.001).

   **Indicates that synovial histopathology scores of IL-1 ra-treated ACLT animals were significantly higher than control animals (p<0.001).

4. Urinary CTXII (uCTXII) levels, adjusted to urinary creatinine in urines collected over a 24 hour period in control animals (n=5) or at 5 weeks post-ACLT treatment with PBS (n=10) or IL-1 ra (n=13). Individual data points are presented and the median value is highlighted with an “X”.

   *Indicates that uCTXII levels in PBS-treated ACLT animals were significantly higher than uCTXII levels in control or IL-1 ra treated ACLT animals (p<0.001).

Figure 2

Impact of IL-1 ra treatment on cartilage and synovial histopathologies and urinary CTXII release following anterior cruciate ligament transection (ACLT) in the rat.

1. Representative Safranin-O (top panel) stained cartilage and Hematoxylin & Eosin (H&E; bottom panel) stained synovium from control animals and animals that underwent ACLT followed by intra-articular injection of PBS on days 1, 3, 5 and 7 (PBS) or IL-1 ra (IL-1 ra). The arrow points to synovial thickening and infiltration of inflammatory cells in the PBS-treated group. Scale = 50 µm.

2. Modified OARSI scores of control (n=5), PBS-treated ACLT animals (n=9) and IL-1 ra-treated ACLT animals (n=9). Individual data points are presented and the median value is highlighted with an “X”.

   *Indicates that OARSI scores of the PBS group was significantly higher than OARSI scores of control or the IL-1 ra groups (p<0.001).

3. Synovial histopathology scores of control (n=5), PBS-treated ACLT animals (n=9) and IL-1 ra-treated ACLT animals (n=9). Individual data points are presented and the median value is highlighted with an “X”.

   *Indicates that synovial histopathology scores of PBS-treated ACLT animals were significantly higher than control and IL-1 ra-treated ACLT animals (p<0.001).

   **Indicates that synovial histopathology scores of IL-1 ra-treated ACLT animals were significantly higher than control animals (p<0.001).

4. Urinary CTXII (uCTXII) levels, adjusted to urinary creatinine in urines collected over a 24 hour period in control animals (n=5) or at 5 weeks post-ACLT treatment with PBS (n=10) or IL-1 ra (n=13). Individual data points are presented and the median value is highlighted with an “X”.

   *Indicates that uCTXII levels in PBS-treated ACLT animals were significantly higher than uCTXII levels in control or IL-1 ra treated ACLT animals (p<0.001).

Figure 3

Impact of treatment with recombinant human lubricin (rhPRG4) and IL-1 ra alone and in combination on chondrocyte apoptosis, lubricin and MMP-13 immunostaining following ACLT and a single intra-articular treatment with IL-1 ra (75 mg/ml; 40 µl), rhPRG4 (200 µg/ml; 40 µl) or IL-1 ra + rhPRG4 (75 mg/ml-200 µg/ml; 40 µl).

1. Representative cleaved caspase-3 (top panel), lubricin (middle panel) and MMP-13 (bottom panel) immunostained articular cartilage from controls and animals that underwent ACLT followed by intra-articular treatment with PBS (PBS), IL-1 ra (IL-1 ra), recombinant lubricin (rhPRG4) and a combination of IL-1 ra and rhPRG4 (IL-1 ra + LUB). Scale = 50 µm.

2. Semi-quantitative analysis of percentage of cleaved caspase-3 positive chondrocytes in controls (n=5) and ACLT animals treated with PBS, IL-1 ra, rhPRG4 or IL-1 ra + rhPRG4 (n=6 in each group). Individual data points are presented and the median value is highlighted with an “X”.

   *Indicates that percentage caspase-3 positive cells in the PBS-treated animals was significantly higher than percentage caspase-3 positive cells in controls (p<0.001), IL-1 ra (p=0.003), rhPRG4 (p=0.001) and IL-1 ra + rhPRG4 (p<0.001).

   **Indicates that percentage caspase-3 positive cells in the IL-1 ra-treated animals was significantly higher than percentage caspase-3 positive cells in controls (p<0.001) and IL-1 ra + rhPRG4 (p=0.039).

Figure 3

Impact of treatment with recombinant human lubricin (rhPRG4) and IL-1 ra alone and in combination on chondrocyte apoptosis, lubricin and MMP-13 immunostaining following ACLT and a single intra-articular treatment with IL-1 ra (75 mg/ml; 40 µl), rhPRG4 (200 µg/ml; 40 µl) or IL-1 ra + rhPRG4 (75 mg/ml-200 µg/ml; 40 µl).

1. Representative cleaved caspase-3 (top panel), lubricin (middle panel) and MMP-13 (bottom panel) immunostained articular cartilage from controls and animals that underwent ACLT followed by intra-articular treatment with PBS (PBS), IL-1 ra (IL-1 ra), recombinant lubricin (rhPRG4) and a combination of IL-1 ra and rhPRG4 (IL-1 ra + LUB). Scale = 50 µm.

2. Semi-quantitative analysis of percentage of cleaved caspase-3 positive chondrocytes in controls (n=5) and ACLT animals treated with PBS, IL-1 ra, rhPRG4 or IL-1 ra + rhPRG4 (n=6 in each group). Individual data points are presented and the median value is highlighted with an “X”.

   *Indicates that percentage caspase-3 positive cells in the PBS-treated animals was significantly higher than percentage caspase-3 positive cells in controls (p<0.001), IL-1 ra (p=0.003), rhPRG4 (p=0.001) and IL-1 ra + rhPRG4 (p<0.001).

   **Indicates that percentage caspase-3 positive cells in the IL-1 ra-treated animals was significantly higher than percentage caspase-3 positive cells in controls (p<0.001) and IL-1 ra + rhPRG4 (p=0.039).