Abstract
Summary
Hyaluronan suppressed lipopolysaccharide-stimulated prostaglandin E2 production via intercellular adhesion molecule-1 through down-regulation of nuclear factor-κB. Administration of hyaluronan into rheumatoid joints may decrease prostaglandin E2 production by activated macrophages, which could result in improvement of arthritic pain.
Introduction
Prostaglandin E2 (PGE2) is one of the key mediators of inflammation in rheumatoid arthritis (RA) joints. Intra-articular injection of high molecular weight hyaluronan (HA) into RA knee joints relieves arthritic pain. Although HA has been shown to inhibit PGE2 production in cytokine-stimulated synovial fibroblasts, it remains unclear how HA suppresses PGE2 production in catabolically activated cells. Furthermore, HA effect on macrophages has rarely been investigated in spite of their contribution to RA joint pathology.
Objectives
This study was aimed to investigate the inhibitory mechanism of HA on lipopolysaccharide (LPS)-stimulated PGE2 in U937 human macrophage culture system.
Methods
With or without pretreatment with one of HA, NS-398, and BAY11-7085, differentiated U937 macrophages were stimulated with LPS. In another set of experiments, the cells were incubated with anti-ICAM-1 antibody or non-specific IgG before pretreatment with HA. PGE2 concentrations of the cell-free supernatants were determined using an enzyme-linked immunosorbent assay. The cell lysates and nuclear extracts were prepared for immunoblot analysis. HA binding to ICAM-1 was evaluated by fluorescence microscopic analysis.
Results
Stimulation of U937 macrophages with LPS enhanced PGE2 production in association with increased protein levels of cyclooxygenase-2 (COX-2). Pretreatment with HA of 2,700 kDa resulted in suppression of LPS-induced COX-2, leading to a decrease in PGE2 production. While LPS activated NF-κB pathway, inhibition studies using BAY11-7085 revealed the requirement of NF-κB for LPS-stimulated PGE2 production. HA down-regulated the phosphorylation and nuclear translocation of NF-κB by LPS. Fluorescence cytochemistry demonstrated that HA bound to ICAM-1 on U937 macrophages. Anti-ICAM-1 antibody reversed the inhibitory effects of HA on LPS-activated PGE2, COX-2, and NF-κB.
Conclusion
These results clearly demonstrated that HA suppressed LPS-stimulated PGE2 production via ICAM-1 through down-regulation of NF-κB. Clinical administration of high molecular weight HA into RA joints may decrease PGE2 production by activated macrophages, which could result in improvement of arthritic pain.
