High mobility group box protein 1 in complex with lipopolysaccharide or IL-1 promotes an increased inflammatory phenotype in synovial fibroblasts

Introduction: In addition to its direct proinflammatory activity, extracellular high mobility group box protein 1 (HMGB1) can strongly enhance the cytokine response evoked by other proinflammatory molecules, such as lipopolysaccharide (LPS), CpG-DNA and IL-1 beta, through the formation of complexes. Extracellular HMGB1 is abundant in arthritic joint tissue where it is suggested to promote inflammation as intra-articular injections of HMGB1 induce synovitis in mice and HMGB1 neutralizing therapy suppresses development of experimental arthritis. The aim of this study was to determine whether HMGB1 in complex with LPS, interleukin (IL)-1 alpha or IL-1 beta has enhancing effects on the production of proinflammatory mediators by rheumatoid arthritis synovial fibroblasts (RASF) and osteoarthritis synovial fibroblasts (OASF). Furthermore, we examined the toll-like receptor (TLR) 4 and IL-1RI requirement for the cytokine-enhancing effects of the investigated HMGB1-ligand complexes. Methods: Synovial fibroblasts obtained from rheumatoid arthritis (RA) and osteoarthritis (OA) patients were stimulated with HMGB1 alone or in complex with LPS, IL-1 alpha or IL-1 beta. Tumour necrosis factor (TNF) production was determined by enzyme-linked immunospot assay (ELISPOT) assessment. Levels of IL-10, IL-1-beta, IL-6 and IL-8 were measured using Cytokine Bead Array and matrix metalloproteinase (MMP) 3 production was determined by ELISA. Results: Stimulation with HMGB1 in complex with LPS, IL-1 alpha or IL-1 beta enhanced production of TNF, IL-6 and IL-8. HMGB1 in complex with IL-1 beta increased MMP production from both RASF and OASF. The cytokine production was inhibited by specific receptor blockade using detoxified LPS or IL-1 receptor antagonist, indicating that the synergistic effects were mediated through the partner ligand-reciprocal receptors TLR4 and IL-1RI, respectively. Conclusions: HMGB1 in complex with LPS, IL-1 alpha or IL-1 beta boosted proinflammatory cytokine- and MMP production in synovial fibroblasts from RA and OA patients. A mechanism for the pathogenic role of HMGB1 in arthritis could thus be through enhancement of inflammatory and destructive mechanisms induced by other proinflammatory mediators present in the arthritic joint.