High mobility group box chromosomal protein 1 (HMGB-1), named for its rapid mobility on electrophoresis gels, is a ubiquitous, nonhistone, chromatin-associated 215–amino acid protein with highly conserved amino acid sequence identity between rodents and humans (1–3). Nuclear HMGB-1 has been identified and studied for a long time as a DNA binding protein. It participates in maintenance of nucleosomal structure and stability and facilitates the binding of transcription factors to their cognate DNA sequences (4). HMGB-1 also has functions in DNA transcription, recombination (5, 6), repair, cell replication, cell migration, and tumor growth (7, 8).
In contrast to its intranuclear role, extracellular HMGB-1 was recently shown to act as a cytokine mediating delayed endotoxin lethality (9) as well as acute lung injury in mice (10). Moreover, high levels of HMGB-1 have been detected in the blood of patients with sepsis (9) and in the synovial fluid of rheumatoid arthritis (RA) patients (11). Proinflammatory mediators, such as tumor necrosis factor α (TNFα) and interleukin-1 (IL-1), can dose-dependently induce the release of HMGB-1 from monocytes and macrophages (9). Furthermore, once released, HMGB-1 itself can activate an additional downstream cascade by stimulating monocytes to produce proinflammatory cytokines and chemokines (e.g., TNFα, IL-1α, IL-1β, macrophage inflammatory protein 1α, IL-6, and IL-8), whereas the cytokine response to HMGB-1 is totally confined to the monocyte/macrophage population (12). Since proinflammatory cytokine release is important in the mediation of arthritis, we reasoned that HMGB-1 might have a direct role in the development of arthritis.
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Our results indicate that the HMGB-1 molecule by itself triggers arthritis in healthy recipients. Also, it is found in joints of patients with RA as well as in animals with adjuvant arthritis (11, 21), and therefore it might play an important pathogenic role in inflammatory joint disease. This proinflammatory effect was not caused by LPS contamination because mice receiving LPS at a dose equivalent to that which could be found in the highest dose of HMGB-1 did not display any signs of arthritis.
Previous studies have shown that inflammatory cytokines, such as TNFα and IL-1, induce the release of HMGB-1 from monocytes and macrophages (12). Furthermore, in vitro studies have revealed that extracellular HMGB-1 itself can activate an additional downstream cascade by stimulating monocyte/macrophages to secrete a subset of proinflammatory cytokines and chemokines. This proinflammatory response to HMGB-1 is highly restricted to the monocyte/macrophage subset since no cytokine production by lymphocytes was seen (12). Indeed, it seems that in the case of HMGB-1–triggered arthritis, the innate immune system plays a major role. The finding strongly supporting this conclusion is that inflammation in the joints occurs at the same frequency and severity in SCID mice lacking T and B cells as in congenic controls. Finally, the scarcity of joint-infiltrating T cells supports this suggestion. In contrast, an abundance of mononuclear Mac-1+ macrophages was found in the inflamed synovial tissue. Moreover, simultaneous depletion of both these cell types diminished the severity as well as the incidence of synovitis, indicating the concomitant action of these 2 major components of innate immunity.
IL-1 is one of the principal mediators of the host response to inflammatory stimuli. The major source of IL-1 is activated mononuclear phagocytes (22). This cytokine, together with TNFα, is considered to play a pivotal role in the development and progression of joint inflammation in rheumatic diseases, such as RA. TNFα is a master cytokine that causes joint swelling, whereas IL-1, the production of which may occur independent of TNFα, has been shown to be more responsible for cartilage and bone destruction (23). In the present study, IL-1R−/− mice did not develop arthritis in response to administration of HMGB-1, suggesting a major role for this macrophage-derived cytokine in HMGB-1–triggered joint inflammation. The reason arthritis developed in 40% of mice of the congenic, IL-1R+/+ strain (C57BL/6) rather than in ∼80% as seen in NMRI and CB17 mice is unknown, but probably reflects differences in background genome and the propensity to develop inflammation following exposure to HMGB-1.
The signaling mechanisms by which HMGB-1 activates cells are not fully understood. HMGB-1 has at least one high-affinity receptor, the receptor for advanced glycation end products (RAGE) (24), which is a multiligand receptor of the immunoglobulin superfamily and is expressed on many cell types, including endothelial cells, mononuclear phagocytes, and smooth muscle cells (25). In this respect, all these cell types are readily found in healthy synovial tissue. In addition, the fact that HMGB-1 increases expression of adhesion molecules and production of chemokines (26) may cause it to serve as an efficient stimulator of the influx of inflammatory cells to the synovial tissue and might contribute to the development of inflammation in the joints.
NF-κB is a critical transcription factor involved in the production of many cytokines and adhesion molecules. In this study, we show that stimulation of spleen cells with HMGB-1 leads to the activation of NF-κB. One needs to keep in mind that the ligation of RAGE by HMGB-1 leads to activation of NF-κB, at least in neural cells (27). Therefore, it remains possible that activation of NF-κB in lymphoid cells found in our experiments might also be mediated via the RAGE receptor pathway. Nevertheless, our results suggest that the proinflammatory effect of HMGB-1 is at least partly mediated by activation of NF-κB.
Previous reports have indicated that HMGB-1 is a late mediator of endotoxin lethality: its release from cultured macrophages was delayed compared with the release of early proinflammatory cytokines (9). The release of HMGB-1 is not only a late response to proinflammatory stimuli, but it also itself provokes a delayed response (12), thereby prolonging and maintaining inflammation. Indeed, our findings indicate that 50% of mice injected with HMGB-1 displayed synovitis up to 28 days following a single injection. The fact that HMGB-1 is not stably associated with the chromosomes (28) and can be released from damaged as well as from necrotic cells (21, 29, 30) might also contribute to the prolongation of the arthritis process because the cells undergoing necrosis at the site of inflammation could serve as an additional source of HMGB-1.
In conclusion, we propose that HMGB-1 is of importance in the pathogenesis of arthritis because it can be found in the inflamed joint and it can induce joint inflammation on its own by activating monocyte/macrophages and inducing the release of the proinflammatory cytokine IL-1 via NF-κB activation. This finding implicates HMGB-1 as a potential target for future therapies.