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Objectives: Binding of mononuclear leukocytes to hyaluronan cable structures is a well-known pathomechanism in several chronic inflammatory diseases, but has not yet described for chronic oral inflammations. The aim of this study was to evaluate if and how binding of mononuclear leukocytes to pathologic hyaluronan cable structures can be induced in human gingival fibroblasts., Material and Methods: Experiments were performed with human gingival fibroblasts and peripheral blood mononuclear cells (PBMCs) from three healthy blood donors. Gingival fibroblasts were stimulated with (1) tunicamycin, (2) polyinosinic/polycytidylic acid (Poly:IC), and (3) lipopolysaccharides (LPS) to simulate (1) ER stress and (2) viral and (3) bacterial infections, respectively. Fibroblasts were then co-incubated with PBMCs, and the number of bound and fluorescently labeled PBMCs was assessed using a fluorescence reader and microscopy. For data analysis, a linear mixed model was used., Results: Hyaluronan-mediated binding of PBMCs to gingival fibroblasts was increased by tunicamycin and Poly(I:C) but not by LPS. Hyaluronidase treatment and co-incubation with hyaluronan transport inhibitors reduced this binding., Conclusions: Results suggest that hyaluronan-mediated binding of blood cells might play a role in oral inflammations. A potential superior role of viruses needs to be confirmed in further clinical studies., Clinical Relevance: The linkage between pathological hyaluronan matrices and oral infections opens up potential applications of hyaluronan transport inhibitors in the treatment of chronic oral inflammations.

Hyaluronan is synthesized within the cytoplasm and exported into the extracellular matrix through the cell membrane of fibroblasts by the MRP5 transporter. In order to meet the law of electroneutrality, a cation is required to neutralize the emerging negative hyaluronan charges. As we previously observed an inhibiting of hyaluronan export by inhibitors of K(+) channels, hyaluronan export was now analysed by simultaneously measuring membrane potential in the presence of drugs. This was done by both hyaluronan import into inside-out vesicles and by inhibition with antisense siRNA. Hyaluronan export from fibroblast was particularly inhibited by glibenclamide, ropivacain and BaCl(2) which all belong to ATP-sensitive inwardly-rectifying K(ir) channel inhibitors. Import of hyaluronan into vesicles was activated by 150 mM KCl and this activation was abolished by ATP. siRNA for the K(+) channels K(ir)3.4 and K(ir)6.2 inhibited hyaluronan export. Collectively, these results indicated that hyaluronan export depends on concurrent K(+) efflux.

The membrane potential is mainly maintained by the K(+) concentration gradient across the cell membrane between the cytosol and the extracellular matrix. Here, we show that extracellular addition of high-molecular weight hyaluronan depolarized the membrane potential of human fibroblasts, human embryonic kidney cells (HEK), and central nervous system neurons in a concentration-dependent manner, whereas digestion of cell surface hyaluronan by hyaluronidase caused hyperpolarization. This effect could not be achieved by other glycosaminoglycans or hyaluronan oligosaccharides, chondroitin sulfate, and heparin which did not affect the membrane potential. Mixtures of high-molecular weight hyaluronan and bovine serum albumin had a larger depolarization effect than expected as the sum of both individual components. The different behavior of high-molecular weight hyaluronan versus hyaluronan oligosaccharides and other glycosaminoglycans can be explained by a Donnan effect combined with a steric exclusion of other molecules from the water solvated chains of high-molecular weight hyaluronan. Depolarization of the plasma membrane by hyaluronan represents an additional pathway of signal transduction to the classical CD44 signal transduction pathway, which links the extracellular matrix to intracellular metabolism.