Hyaluronan inhibits cytokine production by lipopolysaccharide-stimulated U937 macrophages through down-regulation of NF-kappaB 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 (TNFalpha), interleukin (IL)-1beta, and IL-6 were determined by enzyme-linked immunosorbent assay. The phosphorylation of nuclear factor (NF)-kappaB, IkappaBalpha, and mitogen-activated protein kinases (MAPKs) was analyzed by immunoblotting.

RESULTS

LPS stimulated production of TNFalpha, IL-1beta, 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-kappaB and MAPK pathways, whereas HA down-regulated p65 NF-kappaB and IkappaBalpha phosphorylation by LPS without affecting MAPKs. Inhibition studies revealed the requirement of NF-kappaB for LPS-stimulated cytokine production. Anti-ICAM-1 antibody reversed the inhibitory effects of HA on phosphorylation of p65 NF-kappaB and IkappaBalpha.

CONCLUSION

HA of intrinsic molecular weight suppresses LPS-stimulated production of proinflammatory cytokines via ICAM-1 through down-regulation of NF-kappaB and IkappaB. Exogenous HA injected into arthritic joints could act as an anti-NF-kappaB agent by the mechanism demonstrated in the present study.