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Background: Neuroinflammation triggered by infection or trauma is the cause of central nervous system dysfunction. High-mobility group box 1 protein (HMGB1), released from stressed and dying brain cells, is a potent neuroinflammatory mediator. The proinflammatory functions of HMGB1 are tightly regulated by post-translational redox modifications, and we here investigated detailed neuroinflammatory responses induced by the individual redox isoforms. Methods: Male Dark Agouti rats received a stereotactic injection of saline, lipopolysaccharide, disulfide HMGB1, or fully reduced HMGB1, and were accessed for blood-brain barrier modifications using magnetic resonance imaging (MRI) and inflammatory responses by immunohistochemistry. Results and Conclusions: Significant blood-brain barrier disruption appeared 24 h after injection of lipopolysaccharide, disulfide HMGB1, or fully reduced HMGB1 compared to controls, as assessed in post-gadolinium T1-weighted MRI images and confirmed by increased uptake of FITC-conjugated dextran. Immunohistochemistry revealed that both HMGB1 isoforms also induced a local production of IL-1β. Additionally, disulfide HMGB1 increased major histocompatibility complex class II expression and apoptosis. Together, the results demonstrate that extracellular, cerebral HMGB1 causes significant blood-brain barrier disruption in a redox-independent manner and activates several components of neuroinflammation. Blocking HMGB1 might potentially improve clinical outcome in conditions such as stroke and traumatic brain injury.

High mobility group box chromosomal protein 1 (HMGB1) is a DNA-binding nuclear protein that can be released from dying cells and activated myeloid cells. Extracellularly, HMGB1 promotes inflammation. Experimental studies demonstrate HMGB1 to be a pathogenic factor in many inflammatory conditions including arthritis. HMGB1-blocking therapies in arthritis models alleviate disease and confer significant protection against cartilage and bone destruction. So far, the most successful HMGB1-targeted therapies have been demonstrated with HMGB1-specific polyclonal antibodies and with recombinant A box protein, a fragment of HMGB1. The present study is the first to evaluate the potential of a monoclonal anti-HMGB1 antibody (2G7, mouse IgG2b) to ameliorate arthritis. Effects of repeated injections of this antibody have now been studied in two conceptually different models of arthritis: collagen type II–induced arthritis (CIA) in DBA/1 mice and in a spontaneous arthritis disease in mice with combined deficiencies for genes encoding for the enzyme DNase type II and interferon type I receptors. These mice are unable to degrade phagocytozed DNA in macrophages and develop chronic, destructive polyarthritis. Therapeutic intervention in CIA and prophylactic administration of anti-HMGB1 monoclonal antibody (mAb) in the spontaneous arthritis model significantly ameliorated the clinical courses. Anti-HMGB1 mAb therapy also partially prevented joint destruction, as demonstrated by histological examination. The beneficial antiarthritic effects by the anti-HMGB1 mAb in two diverse models of arthritis represent additional proof-of-concept, indicating that HMGB1 may be a valid target molecule to consider for development of future clinical therapy.

Objective. High mobility group box chromosomal protein 1 (HMGB-1) is a DNA binding nuclear protein that can be released from dying cells and activated myeloid cells. Extracellularly, HMGB-1 promotes inflammation. Clinical and experimental studies demonstrate that HMGB-1 is a pathogenic factor in chronic arthritis. Mice with combined gene deficiency for DNase II and IFNRI spontaneously develop chronic, destructive polyarthritis with many features shared with rheumatoid arthritis. DNase II is needed for macrophage degradation of engulfed DNA. The aim of this study was to evaluate a potential pathogenic role of HMGB-1 in this novel murine model. Methods. The course of arthritis, assessed by clinical scoring and histology, was studied in DNase II / IFNRI / mice, in comparison with heterozygous and wild-type mice. Synovial HMGB-1 expression was analyzed by immunohistochemistry. Serum levels of HMGB-1 were determined by Western immunoblotting and enzyme-linked immunosorbent assay (ELISA), and anti–HMGB-1 autoantibodies were detected by ELISA. Macrophage activation was studied by immunostaining for intracellular interleukin-1 and HMGB-1. HMGB-1 was targeted with truncated HMGB-1–derived BoxA protein, acting as a competitive antagonist, with intraperitoneal injections every second day for 5 weeks. Results. DNase II / IFNRI / mice developed symmetric polyarthritis with strong aberrant cytosolic and extracellular HMGB-1 expression in synovial tissue, in contrast to that observed in control animals. Increased serum levels of HMGB-1 and HMGB-1 autoantibodies were recorded in DNase II / IFNRI / mice, both prior to and during the establishment of disease. Systemic HMGB-1–specific blockade significantly ameliorated the clinical disease course, and a protective effect on joint destruction was demonstrated by histologic evaluation. Conclusion. HMGB-1 is involved in the pathogenesis of this spontaneous polyarthritis, and intervention with an HMGB-1 antagonist can mediate beneficial effects.

Retinoids modulate cell proliferation, differentiation and apoptosis in a variety of tumour cells including leukaemia and neuroblastoma, a childhood tumour of the sympathetic nervous system. 13-cis retinoic acid is in clinical use against minimal residual disease in neuroblastoma, where the effect seems to depend on dose, scheduling and tumour mass. Novel retinoids are searched for, to improve potency and lower toxicity. We investigated the effect of the synthetic retinoid Ro 13-6307 on neuroblastoma growth in vitro on SK-N-BE(2) and SH-SY5Y cells. Furthermore, effects on tumour growth and the toxicity profile were investigated in a rat xenograft model. Effects of Ro 13-6307 were compared to 13-cis RA (retinoic acid) in vitro and in vivo. Neuroblastoma cells treated with 1 μM Ro 13-6307 exhibited neuronal differentiation, decreased proliferation and accumulation of cells in G1 phase in at least the same magnitude as 5 μM 13-cis RA. No apoptosis was detected in vitro. Treatment of nude rats with neuroblastoma using Ro 13-6307, 0.12 mg p.o. daily, decreased neuroblastoma growth in vivo, in terms of tumour volume during treatment and tumour weight at sacrifice (p < 0.05). In contrast, Ro 13-6307, 0.08 mg p.o. daily, resulted in no significant reduction in tumour growth. All rats treated with Ro 13-6307 gained less weight than control rats, but they exhibited no other signs of toxicity. The toxicity profile of Ro 13-6307 was similar to what we found with 13-cis RA. Our preclinical results suggest that Ro 13-6307 may be a candidate retinoid for clinical oral therapy of neuroblastoma in children. © 2003 Wiley-Liss, Inc.

Aims: Pathogenic effects of the endogenous inflammatory mediator high mobility group box protein 1 (HMGB1) have been described in several inflammatory diseases. Recent reports have underlined the importance of post-translational modifications (PTMs) in determination of HMGB1 function and release mechanisms. We investigated the occurrence of PTMs of HMGB1 obtained from synovial fluid (SF) of juvenile idiopathic arthritis (JIA) patients. Results: Analyses of 17 JIA patients confirmed high HMGB1 levels in SF. Liquid chromatography tandem mass-spectrometry (LC-MS/MS) analyses of PTMs revealed that total HMGB1 levels were not associated with increased lactate dehydrogenase activity but strongly correlated with nuclear location sequence 2 (NLS2) hyperacetylation, indicating active release of HMGB1. The correlation between total HMGB1 levels and NLS2 hypoacetylation suggests additional, acetylation-independent release mechanisms. Monomethylation of lysine 43 (K43), a proposed neutrophil-specific PTM, was strongly associated with high HMGB1 levels, implying that neutrophils are a source of released HMGB1. Analysis of cysteine redox isoforms, fully reduced HMGB1, disulfide HMGB1, and oxidized HMGB1, revealed that HMGB1 acts as both a chemotactic and a cytokine-inducing mediator. These properties were associated with actively released HMGB1. Innovation: This is the first report that characterizes HMGB1-specific PTMs during a chronic inflammatory condition. Conclusion: HMGB1 in SF from JIA patients is actively released through both acetylation-dependent and -nondependent manners. The presence of various functional HMGB1 redox isoforms confirms the complexity of their pathogenic role during chronic inflammation. Defining HMGB1 release pathways and redox isoforms is critical for the understanding of the contribution of HMGB1 during inflammatory processes. Antioxid. Redox Signal. 24, 605–619.

Neuroblastoma, the most common extracranial solid tumour in children, may undergo spontaneous differentiation or regression, but the majority of metastatic neuroblastomas have poor prognosis despite intensive treatment. Retinoic acid regulates growth and differentiation of neuroblastoma cells in vitro, and has shown activity against human neuroblastomas in vivo. The retinoid 9-cis RA has been reported to induce apoptosis in vitro, and to inhibit the growth of human neuroblastoma xenografts in vivo. However, at given dosage, the treatment with 9-cis RA caused significant toxic side effects. In the present study we investigated the bioavailability of 9-cis RA in rat. In addition, we compared two different dose schedules using 9-cis RA. We found that a lower dose of 9-cis RA (2 mg day−1) was non-toxic, but showed no significant effect on tumour growth. The bioavailability of 9-cis RA in rat was 11% and the elimination half-life (t1/2) was 35 min. Considering the short t1/2, we divided the toxic, but tumour growth effective dose 5 mg dayminus;1 into 2.5 mg p.o. twice daily. This treatment regimen showed no toxicity but only limited effect on tumour growth. Our results suggest that 9-cis RA may only have limited clinical significance for treatment of children with poor prognosis neuroblastoma. © 2001 Cancer Research Campaign http://www.bjcancer.com

The nuclear protein high mobility group box protein 1 (HMGB1) promotes inflammation upon extracellular release. HMGB1 induces proinflammatory cytokine production in macrophages via Toll-like receptor (TLR)-4 signaling in a redox-dependent fashion. Independent of its redox state and endogenous cytokine-inducing ability, HMGB1 can form highly immunostimulatory complexes by interaction with certain proinflammatory mediators. Such complexes have the ability to enhance the induced immune response up to 100-fold, compared with induction by the ligand alone. To clarify the mechanisms for these strong synergistic effects, we studied receptor requirements. Interleukin (IL)-6 production was assessed in supernatants from cultured peritoneal macrophages from mice each deficient in one of the HMGB1 receptors (receptor for advanced glycation end products [RAGE], TLR2 or TLR4) or from wild-type controls. The cultures were stimulated with the TLR4 ligand lipopolysaccaride (LPS), the TLR2 ligand Pam3CysSerLys4 (Pam3CSK4), noninflammatory HMGB1 or each TLR ligand in complex with noninflammatory HMGB1. The activity of the HMGB1-TLR ligand complexes relied on engagement of the same receptor as for the noncomplexed TLR ligand, since HMGB1-LPS complexes used TLR4 and HMGB1-Pam3CSK4 complexes used TLR2. Deletion of any of the intracellular adaptor molecules used by TLR2 (myeloid differentiation factor-88 (MyD88), TIR domain-containing adaptor protein (TIRAP)) or TLR4 (MyD88, TIRAP, TIR domain-containing adaptor-inducing interferon-β (TRIF), TRIF-related adaptor molecule (TRAM)) had similar effects on HMGB1 complex activation compared with noncomplexed LPS or Pam3CSK4. This result implies that the enhancing effects of HMGB1-partner molecule complexes are not regulated by the induction of additional signaling cascades. Elucidating HMGB1 receptor usage in processes where HMGB1 acts alone or in complex with other molecules is essential for the understanding of basic HMGB1 biology and for designing HMGB1-targeted therapies.

A prototypic mechanism for enhancement of inflammation by an endogenous molecule is described. The nuclear protein HMGB1 has previously been demonstrated to act as an alarmin and to promote inflammation upon extracellular release, yet its mode of action is still not well defined. Access to highly purified HMGB1 preparations from prokaryotic and eukaryotic sources enabled studies of activation of human PBMC or synovial fibroblast cultures in response to HMGB1 alone or after binding to cofactors. HMGB1 on its own could not induce detectable IL-6 production. However, strong enhancing effects on induction of proinflammatory cytokine production occurred when the protein associated with each of the separate proinflammatory molecules, rhIL-1β, the TLR4 ligand LPS, the TLR9 ligand CpG-ODN, or the TLR1-TLR2 ligand Pam3CSK4. The bioactivities were recorded in cocultures with preformed HMGB1 complexes but not after sequential or simultaneous addition of HMGB1 and the individual ligands. Individual A-box and B-box domains of HMGB1 had the ability to bind LPS and enhance IL-6 production. Heat denaturation of HMGB1 eliminated this enhancement. Cocultures with HMGB1 and other proinflammatory molecules such as TNF, RANKL, or IL-18 did not induce enhancement. HMGB1 thus acts broadly with many but not all immunostimulatory molecules to amplify their activity in a synergistic manner.

Background and objectives High mobility group box protein 1 (HMGB1) is a prototypic alarmin being released from activated, stressed or dying cells. Extracellular HMGB1 has the ability to induce cell migration as well as cytokine production. Pathogenic effects of HMGB1 have been described in several inflammatory diseases, including arthritis, and HMGB1-specific blockade is beneficial in multiple experimental disease models. Recent reports have underlined the importance of post-translational modifications (PTMs) in determination of HMGB1 function and release mechanisms. We investigated the occurrence of PTMs of HMGB1 obtained from synovial fluid (SF) of juvenile idiopathic arthritis patients (JIA). Materials and methods Synovial fluid was obtained from 17 JIA patients. Total Levels of HMGB1 were determined by ELISA. PTMs of HMGB1 were determined HMGB1-specific immunoprecipitation followed by Liquid chromatography tandem mass-spectrometry (LC-MS/MS). Results Analyses of 17 JIA patients confirmed high HMGB1 levels in SF. Liquid chromatography tandem mass-spectrometry (LC-MS/MS) analyses of PTMs revealed that total HMGB1 levels did not associate with increased LDH activity but strongly correlated with nuclear location sequence 2 (NLS2) hyperacetylation, indicating active release of HMGB1. The correlation between total HMGB1 levels and NLS2 hypoacetylation suggests additional, acetylation-independent release mechanisms. Monomethylation of lysine 43 (K43), a proposed neutrophil-specific PTM, strongly associated with high HMGB1 levels, implying that neutrophils are a source of released HMGB1. Analysis of cysteine redox isoforms: fully reduced HMGB1, disulfide HMGB1 and oxidised HMGB1 revealed that HMGB1 acts as both a chemotactic and a cytokine-inducing mediator. These properties were associated with actively released HMGB1. Conclusions This is the first report that characterises HMGB1-specific PTMs during a chronic inflammatory condition. HMGB1 in SF from JIA patients is actively released through both acetylation- and non-acetylation-dependent manners. The presence of various functional HMGB1 redox isoforms confirms the complexity of its pathogenic role during chronic inflammation. Defining HMGB1 release pathways and redox isoforms is critical for the understanding of the contribution of HMGB1 during inflammatory processes.

Background and objectives The nuclear protein HMGB1 (high mobility group chromosomal protein 1) is secreted by activated macrophages/monocytes and promotes inflammation. Extracellular HMGB1 is present in the synovitis of rheumatoid arthritis (RA) patients and intra-articular injections of recombinant HMGB1 induce arthritis in mice. Thus, the authors hypothesise that HMGB1 represents an endogenous mediator of arthritis as well as a target molecule for successful therapeutic intervention in RA. Polyclonal anti-HMGB1 therapy or recombinant boxA protein works in experimental arthritis models. However, no monoclonal anti-HMGB1 therapy has proven efficient in arthritis models. The aim was to evaluate the antiarthritic potential of a monoclonal anti-HMGB1 antibody (2G7) in two different models of arthritis; collagen-induced arthritis (CIA) in DBA/1 mice and DNase II −/− × IFN-IR −/− mice which spontaneously develop chronic polyarthritis resembling RA. DNase II is specifically needed for macrophage degradation of engulfed DNA. This model is associated with abundant extracellular HMGB1 expression including serum levels of several 100 ng/ml before and during onset of disease. Materials and methods Female and male DNase II −/− × IFN-IR −/− mice, initially 5 weeks old, were treated in 5 weeks every second day with intraperitoneally injections with 100 μg of a mouse monoclonal anti-HMGB1 antibody 2G7 (IgG2b, non-commercial antibody, available upon request) or buffer control. Nine animals were included in each group with an equal sex distribution. Treatment of CIA in female DBA/one mice with anti-HMGB1 antibody 2G7 was initiated when the animal reached an arthritis score of ≤2. The animals were treated for 7 days with daily intraperitoneal injections of 70 μg 2G7 (12 animals) or 70 μg of irrelevant antibody (MOPC-195, mouse IgG2b, 7 animals). The outcome was evaluated by daily clinical scoring. Statistical evaluation was performed using Student9s t test calculated on area under the curve values. Serial joint sections were stained with H&E to evaluate inflammatory cell infiltration or using staining with Safranin O to visualise cartilage damage and bone erosions. Results Systemic HMGB1-specific blockade significantly ameliorated the clinical course in CIA as well as in DNase II −/− × IFN-IR −/− mice, and a protective effect on joint destruction was observed by histological evaluation. Conclusion This study identifies beneficial effect of the HMGB1-specific mAb 2G7 in two different experimental arthritis models. The conserved structure of HMGB1 and the strong cross-reactivity between species of the investigated mAb, has thus enabled them to define a potential mAb candidate for further genetic engineering and clinical testing.

Background and objectives The pro-inflammatory protein HMGB1 is a pathogenic mediator in several inflammatory diseases as demonstrated by beneficial effects of HMGB1-blocking therapy in experimental models including arthritis. High levels of extracellular HMGB1 are found both in experimental models of arthritis and in synovial biopsies from RA patients. Furthermore, intraarticular injection of HMGB1 causes destructive synovitis in mice. The authors and other groups have recently demonstrated that besides its endogenous pro-inflammatory properties, HMGB1 can induce cytokine release by complex formation with various proinflammatory molecules resulting in strong synergy. Direct HMGB1-mediated cytokine induction is mediated via TLR4. Receptor usage by HMGB1-complex formations is however an unresolved issue. The authors set out to test a hypothesis that HMGB1-complexes stimulate cytokine induction via dual receptor interaction. The authors postulated that HMGB1 signals via RAGE and the partner molecule via its reciprocal receptor and that the complexes bring these receptors in close proximity. Methods Recombinant HMGB1 was complexed with low amounts of either the TLR4 ligand LPS or the TLR2 ligand Pam3CSK4. The complexes were used to stimulate intraperitoneal macrophages obtained either from mice deficient in RAGE, TLR2, TLR4, MyD88, TIRAP, TRIF or TRAM or from wild type mice on a C57BL/6 background. Supernatant IL-6 levels assessed by ELISA (R&D Systems) were used as the read-out method. Results HMGB1-LPS and HMGB1-Pam3CSK4 complexes both stimulated IL-6 release in a synergistic mode compared to the individual components. Lack of TLR4 eliminated HMGB1-LPS complex-induced IL-6 production whereas absence of TLR2 eliminated that activated by HMGB1-Pam3CSK4 complexes. Deleting RAGE had however no effect on stimulation by either complex. To study whether the complex stimulation caused changes in the intracellular signaling the authors stimulated cells deficient in the adaptor molecules used by TLR2 (MyD88, TIRAP) or TLR4 (MyD88, TIRAP, TRIF, TRAM) with HMGB1-LPS and HMGB1-Pam3CSK4 complexes. Deletion of any of the adaptor molecules had similar effects on HMGB1 complex activation as compared with non-complexed LPS or Pam 3 CSK 4 . Conclusions Cytokine production induced by LPS or Pam3CSK4 in complex with HMGB1 occurred via their reciprocal receptors. The authors could not demonstrate that HMGB1-RAGE interaction or changes in the signalling pathways was responsible for the synergistic cytokine release. Elucidating HMGB1 receptor usage in processes where HMGB1 acts alone or in complex with other molecules is essential for the understanding of basic HMGB1 biology and for designing HMGB1-targeted therapies.

Background and objectives High mobility group box protein 1 (HMGB1) is a nuclear protein present in all cells. HMGB1 is passively released extracellularly during cell death and, similarly, HMGB1 can be actively secreted from activated cells. Extracellular HMGB1 mediates multiple inflammatory processes. High levels of extracellular HMGB1 have been demonstrated in synovial joints of arthritis patients, intraarticular injection of HMGB1 causes synovitis in mice and neutralisation of HMGB1 by peptide inhibitors or polyclonal antibodies ameliorates experimental arthritis. In this study, we investigated and compared the effects of an HMGB1 peptide inhibitor and a mouse monoclonal antibody (mAb) targeting HMGB1 in an experimental model of arthritis and in acute autoimmune hepatitis. Defining a mAb or peptide inhibitor with ameliorative effects will forward the development of HMGB1 blockade suitable for clinical trials. Methods Collagen induced arthritis (CIA) was induced in DBA/1 mice. Arthritis severity was evaluated by clinical grading of inflammation. Mice with established arthritis were treated every other day with mAb 2G7 or irrIgG and monitored for 7 days. Autoimmune hepatitis was induced by ConcavalinA injection in C57/bl6 mice pretreated with Abox, mAb 2G7 or PBS. After 8 h, sera and liver biopsies were collected. Serum ALT levels, along with HTX staining of liver biopsies were assessed. Liver myeloid peroxidase (MPO) were measured as an indication of neutrophil infiltration. Pro-inflammatory mediator levels were quantified by ELISA. HMGB1 levels were evaluated by ELISA and immunohistochemistry. Results mAb 2G7 treatment significantly ameliorated inflammation in CIA mice and also reduced destruction, as assessed by histological analysis. In mice with autoimmune hepatitis, we recorded an upregulated HMGB1 expression damaged liver lobules. Abox treatment minimised liver damage and mAb 2G7 reduced neutrophil infiltration. Reduced interferon γ and CXCL1 levels were demonstrated in both Abox and mAb 2G7 treated mice. Conclusion By these studies, we have for the first time defined a mAb targeting HMGB1 that effectively reduces inflammation and destruction during CIA. Beneficial effects of this mAb were also demonstrated in a model of autoimmune hepatitis. Furthermore, we could confirm the inhibitory effects of the peptide inhibitor A box, previously demonstrated in CIA, in experimental autoimmune hepatitis. Hence, our current work with humanisation of mAb 2G7 and assessements of its therapeutic effects can be performed in the autoimmune hepatitis model. This allows us to rapidly screen multiple humanised mAb clones and also avoids xenoreactivity effects. Hence, we have facilitated the development of HMGB1 blockade for clinical use.

The proinflammatory protein HMGB1 was recently identified as a mediator of rheumatoid arthritis (RA) and of other inflammatory diseases. This is demonstrated by extracellular HMGB1 expression both in experimental models of arthritis and in synovial biopsies from patients with RA. Furthermore, intra-articular injection of HMGB1 causes destructive synovitis in mice and HMGB1-targeted treatment ameliorates experimental arthritis. Recent data have indicated that HMGB1 alone does not have proinflammatory properties while complexes of HMGB1 and the TLR ligands CpG DNA and lipopolysaccharide …

More than half of the dorsal root ganglion (DRG) neurons are lost by excessive cell death coinciding with precursor proliferation and cell cycle exit in neurotrophin-3 null mutant ( NT-3 −/− ) mice. We find that in the absence of NT-3, sensory precursor cells fail to arrest the cell cycle, override the G1 phase restriction point, and die by apoptosis in S phase, which can be prevented in vivo by a cell cycle blocker. Uncoordinated cell cycle reentry is preceded by a failure of nuclear N-myc downregulation and is paralleled by the activation of the full repertoire of G1 and S phase cell cycle proteins required for cell cycle entry. Our results provide evidence for novel activity of neurotrophins in cell cycle control and point toward an N-myc sensitization to cell death in the nervous system that is under the control of NT-3.