Airway Smooth Muscle Cells Synthesize Hyaluronan “cable” Structures Independent of Heavy Chain Attachment

The covalent association of inter-alphainhibitor (IαI) derived heavy-chains (HCs) with hyaluronan was first described in synovial fluid from arthritic patients and later described as a structural and functional component of hyaluronan “cable” structures produced by many different cells and stimuli. HC-transfer has been shown to be mediated by the protein product of tumor necrosis factor – stimulated gene 6 (TSG-6). Considering the accumulation of hyaluronan in airways following asthmatic attacks and the subsequent infiltration of leukocytes, we sought to characterize HC substitution of hyaluronan “cables” in primary mouse airway smooth muscle cells (MASM) and primary human airway smooth muscle cells (HASM). We found that cells derived from mice lacking TSG-6 had no defect in hyaluronan production or hyaluronanmediated leukocyte adhesion when treated with the viral mimic poly(I:C). Functional hyaluronan “cables” were induced by cycloheximide in the confirmed absence of protein synthesis, with or without simultaneous treatment with poly(I:C). We characterized the species specificity of the antibody other investigators used to describe the HC-hyaluronan complex of hyaluronan “cables” and found minimal affinity to bovine derived HCs in contrast to HCs from mouse and human sera. Thus, we cultured MASM and HASM cells in serum from these three sources and analyzed hyaluronan extracts for HCs and other hyaluronan binding proteins, using parallel cumulus cell – oocyte complex (COC) extracts as positive controls. We conclude that, if hyaluronan “cables” derived from MASM and HASM cells are substituted with HCs, the amount of substitution is significantly below the limit of detection when compared to COC extracts of similar hyaluronan mass.