Hyaluronan inhibits cytokine production by lipopolysaccharide-stimulated U937 macrophages through down-regulation of NF-kappa B via ICAM-1

Objective: This study investigated the inhibitory mechanism of hyaluronan (HA) on lipopolysaccharide (LPS)-stimulated production of proinflammatory cytokines in U937 macrophages. Methods: HA was added to U937 macrophage cultures in the presence of LPS, with or without pretreatment with anti-intercellular adhesion molecule-1 (ICAM-1) antibody. Secreted levels of tumor necrosis factor alpha (TNF alpha), interleukin (IL)-1 beta, and IL-6 were determined by enzyme-linked immunosorbent assay. The phosphorylation of nuclear factor (NF)-kappa B, I kappa B alpha, and mitogen-activated protein kinases (MAPKs) was analyzed by immunoblotting. Results: LPS stimulated production of TNF alpha, IL-1 beta, and IL-6. In contrast to 800 kDa HA, 2700 kDa HA at 1 mg/ml inhibited LPS-induced cytokine production. Anti-ICAM-1 antibody blocked the effects of HA on the LPS actions on U937 cells. LPS activated NF-kappa B and MAPK pathways, whereas HA down-regulated p65 NF-kappa B and I kappa B alpha phosphorylation by LPS without affecting MAPKs. Inhibition studies revealed the requirement of NF-kappa B for LPS-stimulated cytokine production. Anti-ICAM-1 antibody reversed the inhibitory effects of HA on phosphorylation of p65 NF-kappa B and I kappa B alpha. Conclusion: HA of intrinsic molecular weight suppresses LPS-stimulated production of proinflammatory cytokines via ICAM-1 through down-regulation of NF-kappa B and I kappa B. Exogenous HA injected into arthritic joints could act as an anti-NF-kappa B agent by the mechanism demonstrated in the present study.