Mechanism of glycosaminoglycan-mediated bone and joint disease: implications for the mucopolysaccharidoses and other connective tissue diseases

Abstract

We have previously shown that glycosaminoglycan (GAG) storage in animal models of the mucopolysaccharidoses (MPS) leads to inflammation and apoptosis within cartilage. We have now extended these findings to synovial tissue and further explored the mechanism underlying GAG-mediated disease. Analysis of MPS rats, cats, and/or dogs revealed that MPS synovial fibroblasts and fluid displayed elevated expression of numerous inflammatory molecules, including several proteins important for lipopolysaccharide signaling (eg, Toll-like receptor 4 and lipoprotein-binding protein). The expression of tumor necrosis factor, in particular, was elevated up to 50-fold, leading to up-regulation of the osteoclast survival factor, receptor activator of nuclear factor-kappaB ligand, and the appearance of multinucleated osteoclast-like cells in the MPS bone marrow. Treatment of normal synovial fibroblasts with GAGs also led to production of the prosurvival lipid sphingosine-1-phosphate, resulting in enhanced cell proliferation, consistent with the hyperplastic synovial tissue observed in MPS patients. In contrast, GAG treatment of normal chondrocytes led to production of the proapoptotic lipid ceramide, confirming the enhanced cell death we had previously observed in MPS cartilage. These findings have important implications for the pathogenesis and treatment of MPS and have further defined the mechanism of GAG-stimulated disease.

Figure 1

A: Detection of the TLR4, 192-kDa heterodimer; lipopolysaccharide-binding protein (LBP), 57-kDa monomer; adaptor molecule MyD88, 30-kDa monomer; and protein kinase C-α (PKC-α), 82-kDa monomer (as a loading control) by Western blot analysis in normal and MPS VI rat FLSs and chondrocytes. An equal number of cells (7.2 × 10⁵ FLSs and 1.6 × 10⁶ chondrocytes) were used to prepare the extracts, and an equal volume (20 μl) was loaded into each lane. B: TLR4 immunostaining in 12-month-old normal and MPS VI rat articular cartilage. Scale bars = 10 μm.

Figure 2

Effects of dermatan sulfate (DS) on ceramide and S1P levels in rat chondrocytes and FLSs. A: Normal chondrocytes (filled circles) and FLSs (open circles) were grown in standard culture media containing 100 μg/ml of DS for 120 minutes and ceramide levels were determined in cell lysates. B: Normal and MPS VI rat FLSs were grown in standard culture media for 48 hours and S1P levels were determined in cell lysates. Mean (n = 4) ± SEM. *P < 0.001. C: Proliferation studies in FLSs from 1-year-old normal (open boxes) and MPS VI rats (filled boxes). Cells were grown for 48 hours and the proliferation rates were determined. Data represent median values obtained from four independent experiments. MPS VI rat FLSs showed a greater than twofold increase. *P < 0.001.

Figure 3

MIP-1α immunostaining in 12-month-old normal and MPS VII dog bone marrow and synovial membrane. A and C: Normal bone marrow and synovial membrane. B and D: MPS VII bone marrow and synovial membrane showing MIP-1α-positive (arrows) macrophages. Scale bars = 50 μm.

Figure 4

TNF-α and IL-1β levels in synovial fluid from normal and MPS VII dogs, and MPS VI rat FLSs. A: TNF-α and IL-1β concentrations were determined in serum-free FLS-conditioned culture media from MPS VI rats collected after 48 hours. Normal controls are shown by open boxes, and filled boxes represent MPS VI animals. B: Synovial fluid from normal (open boxes) and MPS VII dogs (filled boxes). Mean (n = 4) ± SEM. *P < 0.001.

Figure 5

MMP expression in MPS VI rats. A: MMP-13 immunostaining in age-matched (12-month) normal and MPS VI rat synovial membranes. B: MMP-13 and MMP-1 RNA expression in rat FLSs. PCR analysis for MMP-1 and -13 was performed using mRNA obtained from FLS cultures. C: MMP-13 released into FLS-conditioned, serum-free culture supernatants collected after 48 hours. Mean (n = 4) ± SEM. *P < 0.001. Scale bars = 50 μm.

Figure 6

TRAP-positive multinuclear cells (multinucleated osteoclast-like cells) in bone marrow cultures from MPS VI rats. A: Bone marrow cultures from normal 12-month-old rats. B: Age-matched MPS VI bone marrow cultures showing TRAP-positive multinucleated osteoclast-like cells (arrows). TRAP was used as a marker for osteoclasts. Scale bars = 50 μm.

Figure 7

RANKL expression in 12-month-old rat MPS VI chondrocytes and FLSs. A: PCR analysis was performed using mRNA obtained from chondrocyte and FLS cultures. B: RANKL concentrations were determined in serum-free FLS culture supernatants after 48 hours. Mean (n = 4) ± SEM. *P < 0.001.