HMGB1 in inflammation : Secretion and function

Abstract: Rheumatoid arthritis (RA) is a chronic inflammatory disease characterised by progressive joint destruction. The inflammatory and joint destructive processes in RA are mediated by resident synovial cells and cells recruited from the blood stream and bone marrow. A variety of cytokines, chemokines and proteases contribute to the cartilage and bone destruction. High mobility group box chromosomal protein 1 (HMGB1) was discovered over three decades ago as a transcription-regulating protein. In addition to its nuclear role, HMGB1 expression was detected at the leading edges in motile cells and its active secretion from immune cells was demonstrated. An excessive HMGB1 expression has been recorded in arthritic joints and in synovial fluid from RA patients and HMGB1-blocking therapies have been demonstrated to attenuate the disease course in murine arthritis models, suggesting that HMGB1 is a key player in arthritis. The focus of this thesis work has been to further the understanding of HMGB1 as an inflammatory mediator and its role in arthritis. More specifically, I have studied the induction of HMGB1 secretion from a variety of inflammatory cells, how HMGB1 blockade affects the proinflammatory cytokine pattern in cell cultures and how HMGB1-targeting therapy affects the disease development in collagen-induced arthritis (CIA). Finally, I have also studied the inflammation-inducing capacity of HMGB1 alone and in complex with other proinflammatory molecules. In order to quantify HMGB1 secretion from different cell types an HMGB1-specific ELIspot method was developed. We could demonstrate that HMGB1 was secreted from macrophage/monocytic cells during inflammatory conditions and that the secretion could be inhibited by gold salts and oxaliplatin treatment as detected by ELIspot. We could demonstrate that oxaliplatin-treatment attenuated disease development in murine CIA. A rebound effect with severe and aggressive disease course was demonstrated after one week of treatment which correlated with an excessive extranuclear HMGB1 pattern in the affected joints, indicating an HMGB1-mediated joint inflammation. Furthermore, we have demonstrated in vitro that the proinflammatory activity of HMGB1 is dependent on complex formation between HMGB1 and other inflammation-promoting molecules, such as IL-1beta, LPS and CpG-DNA. Studies using synovial fibroblasts obtained from arthritis patients demonstrated enhanced induction of TNF, IL-6 and IL-8 production and an enchased production of matrix metalloproteinase-1 and -3 when stimulated with HMGB1 in complex with IL-1beta or LPS as compared to either substance alone. Thus, these results suggest that HMGB1 in complex with IL-1beta or LPS can mediate both inflammation and destruction in RA. In conclusion, the studies presented in this thesis strengthen the view of HMGB1 as an inflammation- and destruction-promoting molecule. I have demonstrated HMGB1 secretion from cell types present in the arthritic joint, defined two therapies in clinical use which have the potential to block HMGB1 secretion and verified the anti-rheumatic effect of one of these therapies in murine CIA. By in vitro studies, I have extended the knowledge of the proinflammatory features of HMGB1 and demonstrated both a proinflammatory and prodestructive effect of HMGB1 on synovial fibroblasts. Taken together, the studies in this thesis suggest that HMGB1 is one of the key mediators of arthritic inflammation and joint destruction.