Rheumatoid arthritis (RA) is a chronic joint disease characterized by synovial inflammation and articular tissue destruction. Excessive local production of proinflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1) has been demonstrated to play a central role in the pathogenesis of RA (1). Therapeutic intervention through blockade of these molecules has conferred good clinical protection in many, but not all, patients (1). It is plausible that therapeutic efficacy in rheumatic diseases might be achieved by antagonizing additional proinflammatory cytokines.
High mobility group box chromosomal protein 1 (HMGB-1; previously called HMG-1 or amphoterin) is a widely studied, ubiquitous nuclear protein that is present in eukaryotic cells (2–4). As a nuclear protein, HMGB-1 stabilizes nucleosomes and enables nicking of DNA, which facilitates gene transcription (4, 5). HMGB-1 is a 215–amino acid protein with a uniquely conserved sequence among species. Mouse and rat HMGB-1 are identical; they differ from human HMGB-1 by substitution of only 2 amino acid residues. HMGB-1 has 2 separate and characteristic DNA-binding domains, referred to as HMG A box and B box, respectively, each containing ∼80 amino acid residues arranged in 3 α helices (5).
Recent studies quite unexpectedly revealed that extracellularly released HMGB-1 exerts distinctly different actions from its intranuclear functions (6–9). When released as a cytokine, HMGB-1 is an extremely potent macrophage-stimulating factor (7) and a proinflammatory mediator of inflammation (9, 10). It may signal in part through the receptor for advanced glycation end products (RAGE) (11, 12). The interaction of HMGB-1 with RAGE activates several intracellular signal transduction pathways, including mitogen-activated protein kinases, Cdc42, Rac, and a nuclear translocation of nuclear factor κB (13), the transcription factor classically linked to inflammatory processes. Stimulated monocyte/macrophages actively secrete HMGB-1 through lysosomal exocytosis (14). The molecule is released as a late mediator during acute inflammation and participates in an important way in the pathogenesis of systemic inflammation in sepsis, after the early mediator response has resolved (9). Therapeutic administration of neutralizing antibodies against HMGB-1 confers protection against lethality in experimental sepsis and endotoxemia (6, 15). Stimulated macrophages actively secrete HMGB-1, which in turn, stimulates the production of multiple proinflammatory cytokines. We recently demonstrated that extracellular HMGB-1 has a potent ability to induce TNF and IL-1 synthesis and secretion in monocytes and macrophages (7). HMGB-1 caused a delayed and biphasic release of TNF in comparison with that detected after lipopolysaccharide (LPS) activation.
In addition, HMGB-1 may accumulate extracellularly through a completely different mechanism, since it is passively released from disintegrating nuclei of necrotic cells, in contrast to apoptotic cells. Necrotic HMGB-1–deficient cells have a greatly reduced ability to promote inflammation (16). HMGB-1 might thus be a desired link between unprogrammed cell death and an inflammatory response. The B box domain contains the proinflammatory cytokine functionality of the molecule, whereas the A box region has an antagonistic, antiinflammatory effect with therapeutic potential (17). It was recently demonstrated that purified, truncated HMGB-1 A-box protein dose-dependently inhibits HMGB-1–induced TNF and IL-1β release from activated macrophages. Furthermore, administration of highly purified A-box protein or neutralizing antibodies against HMGB-1 rescued mice from lethal sepsis, even when initial treatment was delayed for 24 hours after the onset of infection (8, 15). These results establish a clinically relevant therapeutic window that is significantly wider than that for other known cytokines (6).
Our research group has developed immunohistochemical staining methods for the detection of HMGB-1 in sections of biopsy specimens (18). These tools enabled the identification of a striking cytoplasmic expression of HMGB-1 in synovial tissue cells in experimental arthritis and RA. In contrast, synovial specimens from normal rodent or human synovial tissue exhibited a strictly nuclear HMGB-1 localization. Immunoblotting analysis of synovial fluid samples from patients with RA further confirmed an abundant extracellular presence of HMGB-1 in inflamed joints (18).
Activated macrophages and necrotic cell death due to synovial hypoxia and complement activation are important features of chronic synovitis. Each of these processes leads to extracellular HMGB-1 release, which might contribute to inflammatory pathogenesis in the joints. This study was designed to evaluate the therapeutic potential of HMGB-1 blockade in established collagen-induced arthritis (CIA).
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We recently reported that the pattern of HMGB-1 expression is altered in synovial biopsy tissues obtained from RA patients and from animals with experimental arthritis, as compared with nonarthritic controls (18), with the proinflammatory mediator being overexpressed cytoplasmically in macrophages. It is also expressed extracellularly in the synovial membrane and is detectable in synovial fluid from patients with RA. Synovial tissue from uninflamed joints mainly displays a cellular HMGB-1 expression that is restricted to the nuclear compartment. When considered with the results described here, it now appears that HMGB-1 may be targeted to therapeutic advantage in a standard preclinical model of arthritis.
The outcome of the present study demonstrates that HMGB-1 blockade therapy with antibodies or antagonizing A-box protein ameliorates CIA in both mice and rats and thus strongly implicates HMGB-1 as a pathogenetic factor in the arthritic process. The molecule has also been shown to be a potent proinflammatory mediator in a number of other conditions, including experimental septicemia (23), endotoxemia (6), and acute lung inflammation (10). Therapies based on neutralizing anti–HMGB-1 antibodies and truncated A-box protein conferred significant clinical protection in all these other disease models, with systemic in vivo administration of A-box protein even significantly increasing the survival rate in a standardized murine sepsis model when treatment was delayed 24 hours from disease onset (15). Additionally, A-box protein dose-dependently inhibited HMGB-1–induced TNF and IL-1 release in cultured macrophages.
We evaluated the effects of HMGB-1 blockade therapy on established disease in experimental arthritis, since this may be more relevant to the design of clinical trials than a pretreatment approach. We did not observe toxicity in the animals treated with either anti–HMGB-1 antibodies or A-box protein. The results of the present protocol using neutralizing anti–HMGB-1 antibodies or purified A-box protein were clearly beneficial, because CIA was ameliorated to a similar extent as that found after anti-TNF antibody treatment of established murine CIA in previous studies (27). HMGB-1 and TNF blockade regimens both led to clinical improvement within 1 or 2 days. Future studies are required to determine whether therapeutic targeting of HMGB-1 or TNF may provide selective opportunities or drawbacks in the treatment of arthritis. Currently, it is not fully understood to what extent HMGB-1 depends on TNF to mediate its proinflammatory function.
The clinical results of the HMGB-1 blockade therapies in CIA are quite promising and are of great conceptual interest. From a practical point of view, it is conceivable that refinement of HMGB-1 antagonists will further improve the results. In the future, neutralizing monoclonal antibodies might lend themselves to new therapeutic trials. The recent recognition of the B-box motif as the cytokine-inducing part of the molecule has been instrumental in generating effective anti–HMGB-1 IgG monoclonal antibodies for future therapeutic studies. The truncated A-box protein used in our studies is a low molecular construct of 80 amino acid residues. The fact that the cytokine-inducing part of the HMGB-1 protein is identical in humans, mice, and rats provides an unusual therapeutic opportunity. HMGB-1–neutralizing antibodies or fusion proteins identified in experimental studies may confer good clinical protection in human disease without any need for modification.
The precise mode of action through which HMGB-1 blockade therapy reduced joint disease cannot be elucidated from the present study and needs further investigation. Immunohistochemical staining assessments indicate inhibited synovial proliferation and pannus formation in animals treated with anti–HMGB-1 antibodies or A-box protein. The joints from these animals also expressed a reduced number of IL-1β–producing cells compared with controls. Efficient neutralization of extracellular HMGB-1 activity would be expected to cause a down-regulation of IL-1β and TNF formation, since HMGB-1 is a potent stimulus of the production of these cytokines (7). Another consequence of HMGB-1 interaction with RAGE concerns its facilitation of tissue penetration. HMGB-1 activates plasminogen (25) and stimulates additional proteolytic enzymes, including several metalloproteinases. The reduced tissue destruction seen in anti–HMGB-1–treated animals may thus be a direct consequence of HMGB-1 neutralization. Taken together, these results depict HMGB-1 as a therapeutic target molecule of considerable interest in human disease.