Biopathological Effects Of β₂-microglobulin Modified With Advanced Glycation End Products On Human Synovial Cells

Background Dialysis-related amyloidosis(DRA) is a serious complication for patients undergoing long-term dialysis. DRA is characterized by amyloid deposition mainly in bone and joint structures, presenting as carnal tunnel syndrome, destructive arthropathy, and subchondral bone erosions and cysts. The pathogenesis of this complication remains unknown. P 2-microglobulin modified with advanced glycation end products (AGE- β 2m) has been demonstrated to be a major constituent of amyloid fibrils. We found that normal human joint synovial cells (HSCs) express the specific AGE receptors, so we hypothesized that biopathological effects of AGE- P 2m on the human joint synovial cells would play a critical role in the pathogenesis of bone and joint injure in DRA. Methods Normal human synovial cells (type A and type B cells) were isolated and cultured in vitro. Binding assay was performed with radiolabeled human serum albumin modified by AGE (AGE-HSA). Specific binding was defined as total binding minus binding in the presence of excess unlabeled AGE-HSA. The normal human type B synovial cells (HSCs) were cultured in vitro with AGE- β 2m. The levels of monocyte chemoattractant protein-1 (MCP- 1) in the supernatants were measured by enzyme-linked immunosorbent assay (ELISA). The expression of mRNA was assessed by Northern blots. Monocyte chemotaxis assays was performed with MCP-1 induced by AGE- β 2m. The levels of TNF- α and IL-1 β in the supernatants were measured by ELISA. Hyaluronan(HA) in the supernatants of HSCs treated with or without AGE- P 2m were detected by radio immunology assay. The molecular weight of HA in the supernatants was -5- assessed by Western blots. Results Specific dose-dependent binding of 1251 AGE HSA to immobilized HSCs was observed with R=4.90±0.75 × 10 /cell,Kd = 1.27±0.19×106M in type A HSCs ,and R= 3.48±0.32×105 /cell, Kd 1.38±0.16×107M in type B HSCs. TNF- α , IL-1β and AGE-HSA upregulated the expression of AGE binding proteins on HSCs. AGE- 13 2m stimulated HSCs to produce MCP-1 and enhanced the expression of MCP-i mRNA with a time- and dose-dependent manner. AGE- β 2m induced effect was inhibited by preincubation of synovial cells with anti-AGE receptor IgG (a -RAGE). MCP-1 induces migration of monocyte in vitro. AGE- β 2m stimulated HSCs to produce TNF- ci and IL-1β with a time- and dose-dependent manner. These effects were inhibited by preincubation of synovial cells with α -RAGE. The 3.7 fold increased HA level was found in the supernatants of cells treated with AGE- 13 2m compared with those treated with medium alone. The molecular weight of HA was 33.0 KD in AGE- β 2m treated supernatants but 37.0 KD in non-treated controls. AGE- β 2m stimulated the production of HA in type B synovial cells with dose- and time-dependent manner. There was no statistical significance in laminin and procollagen type III levels between the supernatants of AGE- β 2m-treated and non-treated cells. Conclusion Normal HSCs express specific AGE receptors, AGE- β 2m stimulates HSCs to produce MCP-1, TNF-α,IL-1β and HA by a pathway mediated by RAGE. These effects of AGE- β 2m on HSCs might be involved in the pathogenesis of bone and joint injure in DRA.