High Mobility Group Box Chromosomal Protein 1 Levels Research Articles

Protective targeting of high mobility group box chromosomal protein 1 in a spontaneous arthritis model

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. 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-1β 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. 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. HMGB-1 is involved in the pathogenesis of this spontaneous polyarthritis, and intervention with an HMGB-1 antagonist can mediate beneficial effects.

NEUTROPHIL ELASTASE INHIBITOR IMPROVES SURVIVAL OF RATS WITH CLINICALLY RELEVANT SEPSIS

Sivelestat sodium hydrate is a selective inhibitor of neutrophil elastase, which is effective in acute lung injury associated with systemic inflammatory response syndrome. However, the effectiveness of sivelestat in sepsis has not been fully examined. In the present study, the effect of sivelestat on severe sepsis in a rat cecal ligation and puncture (CLP) model was investigated. Adult male Sprague-Dawley rats underwent CLP and were randomly divided into two groups: sivelestat-treated group and saline-treated controls. The serum concentrations of several inflammatory mediators were measured. Hematoxylin-eosin staining, and immunohistochemical staining for high-mobility group box chromosomal protein 1 (HMGB1), IL-8, and CD68 were performed on the lungs to assess pathological changes found 12 h after the CLP procedure. Treatment with sivelestat significantly improved the survival rate of the post-CLP septic animals (P = 0.030). Sivelestat also induced a significant reduction in serum IL-1beta (P = 0.038) and IL-10 (P = 0.008) levels in these CLP rats. Serum HMGB1 levels had no significant difference between the sivelestat-treated and the control group. The lungs from sivelestat-treated rats exhibited less severe pathological changes and decreased the numbers of HMGB1, IL-8, and CD68-positive cells (P < 0.001). Sivelestat significantly improved survival rate of rats with clinically relevant sepsis, possibly by attenuating sepsis-induced systemic inflammatory response and lung injury. This may explain the implicated health benefits of sivelestat in reducing morbidity and mortality from sepsis.

Spherical Sulfated Cellulose Adsorbs High-Mobility-Group Box Chromosomal Protein 1 In Vitro and In Vivo

High-mobility-group box chromosomal protein 1 (HMGB1) has recently been identified as a late mediator of various kinds of acute and chronic inflammation. A method for efficiently removing HMGB1 from systemic circulation could be a promising therapy for HMGB1-mediated inflammatory diseases. It is well known that the cationic portion of HMGB1 binds to heparin, which has abundant sulfates in its structure. In this study, we determined whether spherical sulfated cellulose (SC) efficiently adsorbed HMGB1, as well as other inflammatory mediators, in vitro. Then, we investigated the efficacy of hemoperfusion with the SC (SC group) or cellulose beads (control group) at adsorbing endogenous mediators, including HMGB1, in vivo. We have demonstrated that the SC adsorbed significantly larger amounts of HMGB1, interleukin (IL)-4, and IL-8 when compared with cellulose beads, in vitro. Hemoperfusion with the SC for 30 minute, starting 2 hour after an abdominal opening and closure operation, significantly reduced serum HMGB1 levels (p = 0.004) and consistently increased serum IL-10 levels, in vivo. These data suggest the potential benefits of hemoperfusion using the SC in treating HMGB1-mediated inflammatory diseases.

Expression of High Mobility Group Box Chromosomal Protein 1 and Its Modulating Effects on Downstream Cytokines in Systemic Lupus Erythematosus

To compare the expression of high mobility group box chromosomal protein 1 (HMGB1) and the modulating effects on its downstream cytokines in patients with systemic lupus erythematosus (SLE) and healthy controls. HMGB1 concentrations in serum from SLE patients and controls were measured by immunoblot analysis. HMGB1 messenger RNA (mRNA) expression in peripheral blood mononuclear cells (PBMC) was detected by real-time reverse transcription-polymerase chain reaction. Immunofluorescence assay was employed to examine the translocation of HMGB1 in monocytes after endotoxin stimulation. Release of tumor necrosis factor-alpha (TNF-alpha) and interleukin 6 (IL-6) by PBMC after rHMGB1 stimulation was also measured. Serum HMGB1 levels and HMGB1 mRNA expressions in PBMC were elevated in SLE patients compared with controls. A positive correlation was demonstrated between HMGB1 concentrations and SLE Disease Activity Index. There was an inverse correlation between HMGB1 levels and C4 and C3 concentrations in SLE patients. HMGB1 concentrations were higher in patients with vasculitis and myositis. Lipopolysaccharide stimulated a temporarily elevated release of HMGB1 in SLE patients compared with controls. The pattern and localization of HMGB1 staining in monocytes were similar in both groups. After stimulation with rHMGB1, TNF-alpha level decreased but IL-6 level increased in SLE patients compared with controls. Our findings suggest that increased serum levels of HMGB1 in SLE may be associated with lupus disease activity. The altered production of TNF-alpha and IL-6 in response to rHMGB1 stimulation may participate in the disruption of cytokine homeostasis in SLE.

Circulating Levels of a Soluble Form of Receptor for Advanced Glycation End Products and High-Mobility Group Box Chromosomal Protein 1 in Patients With Acute Pancreatitis

To study in patients with acute pancreatitis (AP) the plasma soluble form of the receptor for advanced glycation end products (sRAGE) and high-mobility group box chromosomal protein 1 (HMGB1) levels, followed-up for 12 days after hospitalization, in relation to the occurrence of organ failure and mortality. Thirty-eight patients with severe AP and organ failure (grade 2). A control group (127 patients) consisted of 38 patients with severe AP without organ failure (grade 1) and 89 patients with mild AP (grade 0). Plasma samples for determination of HMGB1 and sRAGE levels were collected on admission and on days 1 and 2, days 3 and 4, and days 7 and 12 after admission. The median of the highest sRAGE levels was higher in grade 2 patients (472 pg/mL; interquartile range [IQR], 259-912) than in grade 0 plus grade 1 patients (349 pg/mL; IQR, 209-544; P = 0.024). Among the patients with detectable HMGB1, the median of the highest HMGB1 levels was 117 ng/mL (IQR, 56-212; n = 24) in grade 2 patients and 87 ng/mL (IQR, 54-161; n = 62) in grade 0 plus grade 1 patients (P = 0.310). We demonstrate that sRAGE level, but not HMGB1 level, is significantly higher in AP patients who develop organ failure than in AP patients without organ failure who recover.

Clinical Value of Serum HMGB1 Levels in Early Detection of Recurrent Squamous Cell Carcinoma of Uterine Cervix: Comparison with Serum SCCA, CYFRA21-1, and CEA Levels

To evaluate the clinical value of serum high mobility group box chromosomal protein 1 (HMGB1) levels in making the early diagnosis of recurrent cervical squamous cell carcinomas (CSCC) and compare it with the value of serum squamous cell carcinoma antigen (SCCA), cytokeratin fragment (CYFRA) 21-1, and carcinoembryonic antigen (CEA) levels. Immunohistochemical staining of tissue from 64 patients with recurrent CSCCs, 72 patients with non-recurrent carcinoma, and 28 healthy participants was performed to determine the expression of HMGB1 protein. The serum levels of the 4 markers in 112 patients with recurrent CSCC, 174 patients with non-recurrent disease, and 128 healthy participants were measured by enzyme-linked immunosorbent assay. The receiver operating characteristic (ROC) curves were constructed and the area under the curve (AUC) was calculated. Higher immunostaining score was found in recurrent CSCC tissue sections than in non-recurrent CSCC sections. Serum HMGB1 levels in patients with recurrent CSCC were significantly higher than in patients with non-recurrent disease and healthy controls. The AUC of HMGB1, SCCA, CYFRA21-1, and CEA was 0.816, 0.768, 0.703, and 0.625, respectively. HMGB1 had the best specificity and positive likelihood ratio (78.0% and 3.25, respectively), whereas SCCA had the best sensitivity and negative likelihood ratio (76.3% and 0.34, respectively). Parallel combined measurements increased the diagnostic sensitivity and serial combination increased the specificity. High serum HMGB1 levels were inversely correlated with disease-free survival (P=0.009, Pearson chi(2) test) and overall survival (P=0.018). HMGB1 was overexpressed in recurrent CSCCs. Serum HMGB1 level could be a useful and specific marker for evaluating the disease recurrence and predicting prognosis in patients with CSCC. Serial combined measurements of serum HMGB1, SCCA, and CYFRA21-1 increased the diagnostic specificity, and parallel combined testing increased the diagnostic sensitivity.

Extracellular high mobility group box chromosomal protein 1 is a coupling factor for hypoxia and inflammation in arthritis

Tissue hypoxia is closely associated with arthritis pathogenesis, and extracellular high mobility group box chromosomal protein 1 (HMGB-1) released from injured cells also has a role in arthritis development. This study was thus undertaken to investigate the hypothesis that extracellular HMGB-1 may be a coupling factor between hypoxia and inflammation in arthritis. Concentrations of tumor necrosis factor alpha, interleukin-6, vascular endothelial growth factor, lactic acid, lactate dehydrogenase, and HMGB-1 were measured in synovial fluid (SF) samples from patients with inflammatory arthropathy (rheumatoid arthritis and pseudogout) and patients with noninflammatory arthropathy (osteoarthritis). The localization of tissue hypoxia and HMGB-1 was also examined in animal models of collagen-induced arthritis (CIA). In cell-based experiments, the effects of hypoxia on HMGB-1 release and its associated cellular events (i.e., protein distribution and cell viability) were studied. In SF samples from patients with HMGB-1-associated inflammatory arthropathy (i.e., samples with HMGB-1 levels >2 SD above the mean level in samples from patients with noninflammatory arthropathy), concentrations of HMGB-1 were significantly correlated with those of lactic acid, a marker of tissue hypoxia. In CIA models in which the pathologic phenotype could be attenuated by HMGB-1 neutralization, colocalization of HMGB-1 with tissue hypoxia in arthritis lesions was also observed. In cell-based experiments, hypoxia induced significantly increased levels of extracellular HMGB-1 by the cellular processes of secretion and/or apoptosis-associated release, which was much more prominent than the protein release in necrotic cell injury potentiated by oxidative stress. These findings indicate that tissue hypoxia and its resultant extracellular HMGB-1 might play an important role in the development of arthritis.

Pivotal Advance: HMGB1 expression in active lesions of human and experimental multiple sclerosis

Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating disease of the CNS, most frequently starting with a series of bouts, each followed by complete remission and then a secondary, progressive phase during which the neurological deficit increases steadily. The underlying molecular mechanisms responsible for disease progression are still unclear. Herein, we demonstrate that high mobility group box chromosomal protein 1 (HMGB1), a DNA-binding protein with proinflammatory properties, is evident in active lesions of MS and experimental autoimmune encephalomyelitis (EAE) and that HMGB1 levels correlate with active inflammation. Furthermore, the expression of the innate HMGB1 receptors--receptor for advanced glycation end products, TLR2, and TLR4--was also highly increased in MS and rodent EAE. Additionally, in vitro activation of rodent CNS-derived microglia and bone marrow-derived macrophages demonstrated that microglia were equally as capable as macrophages of translocating HMGB1 following LPS/IFN-gamma stimulation. Significant expression of HMGB1 and its receptors on accumulating activated macrophages and resident microglia may thus provide a positive feedback loop that amplifies the inflammatory response during MS and EAE pathogenesis.

Elevated levels of high mobility group box chromosomal protein-1 (HMGB-1) in sera from patients with severe bacterial pneumonia coinfected with influenza virus

Plasma levels of high mobility group box chromosomal protein-1 (HMGB-1), as well as of other inflammatory molecules such as interleukin-6 (IL-6), regulated on activation normal T-cell expressed and secreted (RANTES), and soluble intercellular adhesion molecule-1 (sICAM-1), were determined in patients with bacterial pneumonia coinfected with influenza virus. HMGB-1 levels were significantly elevated in these patients compared to patients undergoing mild bacterial pneumonia alone (p < 0.01). Among cases of coinfection, we found a significant correlation between the concentration of HMGB-1 and white blood cell counts (p < 0.05, r = 0.612). Levels of IL-6 were also higher in these patients than in patients with bacterial pneumonia alone (p < 0.05), despite similar levels of RANTES and sICAM-1 in the 2 groups. These data suggest that HMGB-1 is involved in the pathogenesis of severe bacterial pneumonia coinfected with influenza virus.

Significant Increase of Serum High-Mobility Group Box Chromosomal Protein 1 Levels in Patients With Severe Acute Pancreatitis

Multiple organ failure because of systemic inflammatory response in the early phase and sepsis in the late phase is the main contributor to high mortality in severe acute pancreatitis (SAP). High-mobility group box chromosomal protein 1 (HMGB1) was recently identified as a potent proinflammatory mediator and increases in various pathological conditions such as sepsis. The aim of this study was to investigate contributions of HMGB1 in SAP. We measured serum HMGB1 concentrations by an enzyme-linked immunosorbent assay in 45 patients with SAP at the time of admission. Furthermore, relationship between their serum HMGB1 levels and clinical factors was analyzed. The mean value of serum HMGB1 levels was significantly higher in patients with SAP (5.4 +/- 1.3 ng/mL) than that in healthy volunteers (1.7 +/- 0.3 ng/mL). Serum HMGB1 levels were significantly positively correlated with the Japanese severity score and Glasgow score. Serum HMGB1 levels were significantly positively correlated with lactate dehydrogenase, C-reactive protein, and total bilirubin. The HMGB1 levels were higher in patients with organ dysfunction and infection during the clinical course. The HMGB1 levels in nonsurvivors were higher than those in survivors. Serum HMGB1 levels gradually declined after the admission. Serum HMGB1 levels were significantly increased in patients with SAP and were correlated with disease severity. These results suggest that HMGB1 may act as a key mediator for inflammation and organ failure in SAP.

Serum concentrations of high-mobility group box chromosomal protein 1 before and after exposure to the surgical stress of thoracic esophagectomy: a predictor of clinical course after surgery?

High-mobility group box chromosomal protein 1 (HMGB-1) has recently been shown as an important late mediator of endotoxin shock, intra-abdominal sepsis, and acute lung injury. However, its role in the systemic inflammatory response syndrome after major surgical stress, which may lead to multiple organ dysfunction syndrome, has not been thoroughly investigated. We hypothesized that serum HMGB-1 participates in the pathogenesis of postoperative organ system dysfunction after exposure to major surgical stress. A prospective clinical study was performed to consecutive patients (n = 24) with carcinoma of the thoracic esophagus who underwent transthoracic esophagectomy with three field lymph node resection between 1998 and 2003 at Keio University Hospital, Japan. Serum HMGB-1 concentrations were measured by enzyme-linked immunosorbent assay. Preoperative serum HMGB-1 levels correlated with postoperative duration of SIRS, mechanical ventilation, and intensive care unit stay. Three of the 24 patients had serious postoperative complications: sepsis in two, and acute lung injury in one. Serum HMGB-1 levels in patients without complications increased within the first 24 h postoperatively, remained high during postoperative days 2-3, and then decreased gradually by postoperative day 7. In patients with serious complications, serum HMGB-1 was significantly higher than that found in patients without postoperative complications at every time point except postoperative day 2. Preoperative serum HMGB-1 concentration seems to be an important predictor of the postoperative clinical course. Transthoracic esophagectomy induces an increase in HMGB-1 in serum even in patients without complications. Postoperative serum HMGB-1 concentrations were higher in patients who developed complications, and may be a predictive marker for complications in this setting.

Plasma Levels of High Mobility Group Box Protein in Patients with Organ Failure, Disseminated Intravascular Coagulation or Poor Outcome.

High Mobility Group Box chromosomal protein 1 (HMGB1) is a nuclear DNA-binding protein acting as a proinflammatory cytokine when released in the extracellular space from necrotic cells, activated macrophages and dendritic cells. HMGB1 acts on a specific receptor, RAGE (receptor for advanced glycation end-products), and induces prolonged inflammation, organ failure, septicaemia and death. The aim of the study was to determine the diagnostic value of plasma HMGB1 concentration and its role in the development of organ failure in patients with disseminated intravascular coagulation (DIC).Plasma HMGB-1 levels were measured in patients with suspected DIC by ELISA and their relationships with DIC, organ failure and clinical outcome were determined. The study group included 201 patients suspected of having DIC (79 females and 122 males, age; 52.1±16.5 years, mean±SD).The study group represented all such patients admitted to the Second Department of Internal Medicine and Intensive Care Unit (ICU) of Mie University Hospital between January 1, 2002 and December 31, 2003. The underlying diseases were infectious diseases (n=40), hematopoietic diseases (n=58), solid cancers (n=16), trauma (n=21), aneurysm (n=12), autoimmune diseases (n=8), liver diseases (n=7), post-operation (n=32) and other diseases (n=7).Results: Plasma HMGB1 was below the detection limit in normal subjects, but moderately elevated in patients with infectious diseases (4.54 ± 8.18 ng/ml, mean±SD), malignancies (2.15 ± 5.34 ng/ml), and traumas (6.47 ± 13.13 ng/ml). DIC was associated with significantly high plasma HMGB1 (14.05 ± 12.56 ng/ml) in these patients. The highest HMGBI levels were in patients with organ failure (8.29 ± 10.99 ng/ml) and non-survivors (16.58 ± 11.01 ng/ml). HMGB1 plasma levels correlated with the DIC score and sepsis-related organ failure assessment (SOFA) score. Plasma levels of HMGB1 were significantly higher in patients with PAI-I of >50 ng/ml (3.66 ± 6.36 ng/ml) than in those with PAI-I <50 ng/ml (1.38 ± 2.97 ng/ml) (p< 0.05) but there was no significant difference in HMGB1 levels between patients with TNF-a of >1.5 (3.04 ± 5,56 ng/ml) and those with less than 1.5 pg/ml (2.44 ± 5.36 ng/ml).Our data suggest that HMGB-1 is a potentially suitable prognostic marker of OF or DIC.

HMGB1 is a potent trigger of arthritis.

Rheumatoid arthritis is a chronic inflammatory disease characterized by synovial inflammation and structural damage of joints. Although the cause of rheumatoid arthritis (RA) remains unknown, the excessive production of proinflammatory cytokines such as tumour necrosis factor (TNF) and interleukin-1 (IL-1) by intra-articular macrophages occupies a critical pathogenic role in the development and progression of the disease. High mobility group box chromosomal protein 1 (HMGB1) is a recently identified mediator of interest in human and experimental arthritides. HMGB1 can either be actively secreted from macrophages or passively released from necrotic cells of all kinds. Activated macrophages and unprogrammed cell death caused by ischaemia or activated complement are all prominent features of chronic arthritis, contributing to the persistent synovial inflammation. HMGB1 is cytoplasmically and extracellularly overexpressed in inflammatory synovial tissue in human RA as well as experimental collagen-induced arthritis. Elevated levels of HMGB1 are also present in synovial fluid samples from RA patients. Synovial tissue from rats with experimental arthritis exhibits aberrant deposition of HMGB1 preceding the onset of clinical signs of arthritis, and the expression becomes prominent after the onset of clinical disease. The synovial levels of HMGB1 are comparable with those of TNF and IL-1beta at the peak of manifest disease. HMGB1-targeted intervention with either neutralizing antibodies or the antagonistic A box domain of HMGB1 ameliorates collagen-induced arthritis both in mice and rats, and inhibits the local overexpression of IL-1beta in the joints. It is thus conceivable that therapeutic HMGB1 blockade may contribute to future treatment of human chronic arthritis.

Limitation of Polymyxin B on Suppression of Endotoxin Shock Induced by Salmonella Infection in Mice

The protective effects of an antibiotic polymyxin B (PLB), having lipopolysaccharide (LPS)-binding activity, on infection-induced endotoxin shock in mice were investigated. Infection with 10(8) colony forming units of an attenuated Salmonella typhimurium aroA strain caused lethal endotoxin shock to ddY mice. Treatment with PLB 1 h post infection (p.i.) resulted in significant reduction of mortality and bacterial numbers in livers. In addition, treatment with PLB 1 h p.i. resulted in a transient increase at the early stage and gradual decline in plasma LPS levels. Although plasma levels of sCD14 and high mobility group box chromosomal protein-1 (HMGB-1) increased according with progression of infection, increases in plasma levels of sCD14 and HMGB-1 were downregulated by treatment with PLB 1 h p.i. However, the lethal shock was not blocked by treatment with anti-CD14 monoclonal antibody at 3 h and 6 h p.i. Interestingly, administration of PLB 6 h p.i. did not show any protective activities, indicating that a time window for effective PLB action is present.

High mobility group box chromosomal protein 1: a novel proinflammatory mediator in synovitis.

High mobility group box chromosomal protein 1 (HMGB-1) is a ubiquitous chromatin component expressed in nucleated mammalian cells. It has recently and unexpectedly been demonstrated that stimulated live mononuclear phagocytes secrete HMGB-1, which then acts as a potent factor that causes inflammation and protease activation. Macrophages play pivotal roles in the pathogenesis of arthritis. The aim of this study was to determine whether synovial macrophage expression of HMGB-1 is altered in human and experimental synovitis. Intraarticular tissue specimens were obtained from healthy Lewis rats, Lewis rats with Mycobacterium tuberculosis-induced adjuvant arthritis, and from patients with rheumatoid arthritis (RA). Specimens were immunohistochemically stained for cellular HMGB-1. Extracellular HMGB-1 levels were assessed in synovial fluid samples from RA patients by Western blotting. Immunostaining of specimens from normal rats showed that HMGB-1 was primarily confined to the nucleus of synoviocytes and chondrocytes, with occasional cytoplasmic staining and no extracellular matrix deposition. In contrast, inflammatory synovial tissue from rats with experimental arthritis as well as from humans with RA showed a distinctly different HMGB-1 staining pattern. Nuclear HMGB-1 expression was accompanied by a cytoplasmic staining in many mononuclear cells, with a macrophage-like appearance and an extracellular matrix deposition. Analysis of synovial fluid samples from RA patients further confirmed the extracellular presence of HMGB-1; 14 of 15 samples had HMGB-1 concentrations of 1.8-10.4 microg/ml. The proinflammatory mediator HMGB-1 was abundantly expressed as a nuclear, cytoplasmic, and extracellular component in synovial tissues from RA patients and from rats with experimental arthritis. These findings suggest a pathogenetic role for HMGB-1 in synovitis and indicate a new potential therapeutic target molecule.