TSG-6 and calcium ions are essential for the coupling of inter-alpha-trypsin inhibitor to hyaluronan in human synovial fluid

Abstract

Objective: To investigate the role of tumor necrosis factor stimulated gene 6 (TSG-6) and metal ions in the coupling of inter-α-trypsin inhibitor (ITI) to hyaluronan in human synovial fluid. Design: The concentration of ITI heavy chains bound to hyaluronan was determined by a two-step electrophoretic technique. Synovial fluid, TSG-6 depleted synovial fluid and metal chelated synovial fluid were tested for their ability to support the coupling of ITI heavy chains to hyaluronan. Results: When synovial fluid was mixed with an ITI-source (serum or purified ITI), coupling of ITI heavy chains to hyaluronan took place. TSG-6 immunodepleated synovial fluid lost the coupling activity, but addition of recombinant TSG-6 restored the activity. EDTA inhibited the coupling activity, but combinations of the metal-ion chelators Mg-EGTA and Ca-EGTA demonstrated, that Ca ++is essential for the coupling of ITI heavy chains to hyaluronan. Conclusions: Tumor necrosis factor stimulated gene 6 (TSG-6) and calcium ions are both essential for the coupling of inter-α-trypsin inhibitor to hyaluronan in human synovial fluid.