[show abstract][hide abstract] ABSTRACT: High mobility group box 1 protein (HMGB1) was identified as a mediator of endotoxin lethality. We previously reported that thrombomodulin (TM), an endothelial thrombin-binding protein, bound to HMGB1, thereby protecting mice from lethal endotoxemia. However, the fate of HMGB1 bound to TM remains to be elucidated.
TM enhanced thrombin-mediated cleavage of HMGB1. N-terminal amino acid sequence analysis of the HMGB1 degradation product demonstrated that thrombin cleaved HMGB1 at the Arg10-Gly11 bond. Concomitant with the cleavage of the N-terminal domain of HMGB1, proinflammatory activity of HMGB1 was significantly decreased (P<0.01). HMGB1 degradation products were detected in the serum of endotoxemic mice and in the plasma of septic patients with disseminated intravascular coagulation (DIC), indicating that HMGB1 could be degraded under conditions in which proteases were activated in the systemic circulation.
TM not only binds to HMGB1 but also aids the proteolytic cleavage of HMGB1 by thrombin. These findings highlight the novel antiinflammatory role of TM, in which thrombin-TM complexes degrade HMGB1 to a less proinflammatory form.
[show abstract][hide abstract] ABSTRACT: The high mobility group box chromosomal protein 1 (HMGB1), a nuclear DNA-binding protein, has recently been recognized as a new proinflammatory cytokine. The purpose of this study was to examine the significance of HMGB1 in patients with renal diseases.
HMGB1 concentrations in sera were measured by enzyme-linked immunosorbent assay, and antibodies against HMGB1 were examined by Western blotting in patients who underwent renal biopsies and in healthy controls. Immunohistochemistry for HMGB1 was also performed.
Serum HMGB1 was more likely to be positive in patients who underwent renal biopsies as compared with the controls. Patients with anti-neutrophil cytoplasmic antibody-related glomerulonephritis (ANCA-GN) and those with Henoch-Schonlein purpura nephritis showed a significantly higher tendency to be HMGB1 positive. The presence of anti-HMGB1 antibody was not associated with the presence of serum HMGB1. Immunohistochemistry revealed that HMGB1 was expressed in mononuclear cells in the interstitium or in the glomeruli of some patients with ANCA-GN or IgA nephropathy (IgAN). Subanalysis demonstrated that among patients with IgAN, those who had crescent formation showed a higher tendency to be HMGB1 positive than those who did not.
HMGB1 was expressed in the sera of patients with renal diseases who underwent renal biopsies, especially among those who had vasculitis including ANCA-GN, Henoch-Schonlein purpura nephritis, and IgAN with glomerular crescents.
Nephron Clinical Practice 02/2008; 108(3):c194-201. · 1.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: The aim of the present study was to examine whether neutrophil and its elastase activity played consequential roles in the progression of gut barrier dysfunction during acute alveolar hypoxia by using a specific neutrophil elastase inhibitor, sivelestat. With our institutional approval, 20 male rabbits (weight, 2.0-2.5 kg) were randomly allocated into two groups: control (n = 11) or sivelestat group (n = 9; bolus, 10 mg/kg, followed by 10 mg/kg per hour). At 4 h of alveolar hypoxia exposure (fraction of inspired oxygen, 0.10) under mechanical ventilation, the white blood cell counts and their function to produce oxygen radicals were measured. Intestinal permeability and myeloperoxidase activity were also assessed concurrently with the examination of histological changes of gut mucosa. The examination of sham animals (n = 4) exposed to normoxia was performed under the same study protocol. The circulating leukocyte counts and the neutrophil chemiluminescence were not different between the groups, whereas the neutrophil elastase activity was significantly increased in the control but not in the sivelestat and sham groups. Permeability, leukocyte accumulation, and myeloperoxidase activity of ileal wall in the control group were significantly elevated, accompanied by apparent destruction of gut mucosa compared with the sivelestat group (P < 0.05). Despite no significant differences in systemic inflammatory responses, the neutrophil elastase activity is a key element in the progression of functional and structural injury of gut mucosa during acute alveolar hypoxia.
[show abstract][hide abstract] ABSTRACT: High mobility group box 1 (HMGB1) exhibits unique biochemical functions as a biologically intrinsic requisite factor and as a toxin. As such, it is imperative to understand the mechanism by which these seemingly and diametrically opposed functions are exerted. To effectively discriminate these actions is important to accurately and precisely determine the concentration of HMGB1 in biological samples. Research in this fascinating field, however, has been lacking due to the absence of a simple analytical system for HMGB1 that can be adapted for large sample numbers. In this report, we review the physiological and pathological significance of HMGB1 and describe the development of an assay method for this pleiotropic protein.
[show abstract][hide abstract] ABSTRACT: High-sensitivity sandwich ELISA methods have been developed using chemiluminescent substrates. HMGB1 (high mobility group box 1) protein has been shown to play a critical role in several inflammatory diseases and it may be involved in the development of atherosclerosis.
Anti-human HMGB1 monoclonal antibodies and anti-peptide polyclonal antibodies against the peptide sequence (KPDAAKKGVVKAEK) with high antigenicity and different from the sequence of HMGB2 were developed, and the antibodies were used to construct sandwich ELISA methods with a chromogenic substrate (TMBZ) and a chemiluminescent substrate (PS-atto). Highly purified human HMGB1 was used as a standard material and high-sensitivity CRP was measured to compare with HMGB1.
The analytical characteristics of the ELISA method we developed were validated inter-assay and intra-assay CVs were <10%, and the detection limit was 0.3 microg/l by the chemiluminescent method and 1 microg/l with the chromogenic substrates. HMGB1 was detected in the serum of patients with acute coronary syndrome (ACS). When a cut-off of 0.6 microg/l HMGB1 upon admission to the intensive care unit (ICU) was used, the risk of developing an acute cardiac event within 1 month after discharge of ACS patients with an abnormal HMGB1 was significantly higher than for the patients with normal values (P<0.0001). The usefulness of HMGB1 as an acute prognostic marker was suggested.
The assay is easy to perform and suitable for use in the hospital laboratory and for screening large populations. HMGB1 is detectable in the serum of ACS patients and that the serum concentration of HMGB1 may be a prognostic indicator in ACS patients.
[show abstract][hide abstract] ABSTRACT: Proinflammatory cytokines play an important role in ventilator-induced lung injury (VILI). High-mobility group box-1 (HMGB1) is a macrophage-derived proinflammatory cytokine that can cause lung injury.
This study tested the hypothesis that HMGB1 is released in intact lungs ventilated with large Vt. A second objective was to identify the source of HMGB1. A third objective was to examine the effects of blocking HMGB1 on the subsequent development of VILI.
Bronchoalveolar lavage fluid (BALF) and lung tissues were obtained from rabbits mechanically ventilated for 4 h with a small (8 ml/kg) versus a large (30 ml/kg) Vt. BALF was also obtained from rabbits with intratracheal instillation of anti-HMGB1 antibody before the initiation of large Vt ventilation.
The concentrations of HMGB1 in BALF were fivefold higher in the large than in the small Vt group. Immunohistochemistry and immunofluorescence studies revealed expression of HMGB1 in the cytoplasm of macrophages and neutrophils in lungs ventilated with large Vt. Blocking HMGB1 improved oxygenation, limited microvascular permeability and neutrophil influx into the alveolar lumen, and decreased concentrations of tumor necrosis factor-alpha in BALF.
These observations suggest that HMGB1 could be one of the deteriorating factors in the development of VILI.
American Journal of Respiratory and Critical Care Medicine 09/2006; 174(4):400-7. · 11.04 Impact Factor
[show abstract][hide abstract] ABSTRACT: High-mobility group box chromosomal protein 1 (HMGB1) has recently been shown to be an important late mediator of endotoxin shock, intraabdominal sepsis, and acute lung injury, and a promising therapeutic target of severe sepsis. We sought to investigate the effect of antibodies to HMGB1 on severe sepsis in a rat cecal ligation and puncture (CLP) model.
Adult male Sprague-Dawley rats underwent CLP and then were randomly divided into two groups: treatment with anti-HMGB1 polyclonal antibodies, and non-immune IgG-treated controls. The serum HMGB1 concentrations were measured at ten time points (preoperatively, and postoperatively at 4, 8, 20, 32, and 48 h and at 3, 4, 5, and 6 days). Hematoxylin-eosin staining, elastica-Masson staining, and immunohistochemical staining for HMGB1 were performed on the cecum and the lung to assess pathological changes 24 h after the CLP procedure.
Treatment with anti-HMGB1 antibodies significantly increased survival [55% (anti-HMGB1) vs. 9% (controls); P< 0.01]. The serum HMGB1 concentrations at postoperative hours 20 and 32 of the anti-HMGB1 antibody-treated animals were significantly lower than those of the controls (P < 0.05). Treatment with anti-HMGB1 antibodies markedly diminished the pathological changes and the number of HMGB1-positive cells in the cecum and the lung.
The present study demonstrates that anti-HMGB1 antibodies are effective in the treatment of severe sepsis in a rat model, thereby supporting the relevance of HMGB1 eradication therapy for severe sepsis.
World Journal of Surgery 09/2006; 30(9):1755-62. · 2.23 Impact Factor
[show abstract][hide abstract] ABSTRACT: High mobility group box 1 (HMGB1), originally described as a DNA-binding protein, can also be released extracellularly and functions as a late mediator of inflammatory responses. Although recent reports have indicated that the receptor for advanced glycation end products (RAGE) as well as Toll-like receptor (TLR)2 and TLR4 are involved in cellular activation by HMGB1, there has been little evidence of direct association between HMGB1 and these receptors. To examine this issue, we used fluorescence resonance energy transfer (FRET) and immunoprecipitation to directly investigate cell surface interactions of HMGB1 with TLR2, TLR4, and RAGE. FRET images in RAW264.7 macrophages demonstrated association of HMGB1 with TLR2 and TLR4 but not RAGE. Transient transfections into human embryonic kidney-293 cells showed that HMGB1 induced cellular activation and NF-kappaB-dependent transcription through TLR2 or TLR4 but not RAGE. Coimmunoprecipitation also found interaction between HMGB1 and TLR2 as well as TLR4, but not with RAGE. These studies provide the first direct evidence that HMGB1 can interact with both TLR2 and TLR4 and also supply an explanation for the ability of HMGB1 to induce cellular activation and generate inflammatory responses that are similar to those initiated by LPS.
[show abstract][hide abstract] ABSTRACT: 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 and their relationships with DIC, organ failure and clinical outcome were determined. The study took place at the intensive care facility, Mie University School of Medicine and comprised 201 patients with suspected DIC. 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 HMGB1 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. In conclusion, our data suggest that HMGB-1 is a potentially suitable prognostic marker of OF or DIC.
Thrombosis and Haemostasis 12/2005; 94(5):975-9. · 6.09 Impact Factor
[show abstract][hide abstract] ABSTRACT: Thrombomodulin (TM) is an endothelial anticoagulant cofactor that promotes thrombin-mediated formation of activated protein C (APC). We have found that the N-terminal lectin-like domain (D1) of TM has unique antiinflammatory properties. TM, via D1, binds high-mobility group-B1 DNA-binding protein (HMGB1), a factor closely associated with necrotic cell damage following its release from the nucleus, thereby preventing in vitro leukocyte activation, in vivo UV irradiation-induced cutaneous inflammation, and in vivo lipopolysaccharide-induced lethality. Our data also demonstrate antiinflammatory properties of a peptide spanning D1 of TM and suggest its therapeutic potential. These findings highlight a novel mechanism, i.e., sequestration of mediators, through which an endothelial cofactor, TM, suppresses inflammation quite distinctly from its anticoagulant cofactor activity, thereby preventing the interaction of these mediators with cell surface receptors on effector cells in the vasculature.
Journal of Clinical Investigation 06/2005; 115(5):1267-74. · 12.81 Impact Factor
[show abstract][hide abstract] ABSTRACT: High mobility group box 1 (HMGB1) is a novel late mediator of inflammatory responses that contributes to endotoxin-induced acute lung injury and sepsis-associated lethality. Although acute lung injury is a frequent complication of severe blood loss, the contribution of HMGB1 to organ system dysfunction in this setting has not been investigated. In this study, HMGB1 was detected in pulmonary endothelial cells and macrophages under baseline conditions. After hemorrhage, in addition to positively staining endothelial cells and macrophages, neutrophils expressing HMGB1 were present in the lungs. HMGB1 expression in the lung was found to be increased within 4 h of hemorrhage and then remained elevated for more than 72 h after blood loss. Neutrophils appeared to contribute to the increase in posthemorrhage pulmonary HMGB1 expression since no change in lung HMGB1 levels was found after hemorrhage in mice made neutropenic with cyclophosphamide. Plasma concentrations of HMGB1 also increased after hemorrhage. Blockade of HMGB1 by administration of anti-HMGB1 antibodies prevented hemorrhage-induced increases in nuclear translocation of NF-kappa B in the lungs and pulmonary levels of proinflammatory cytokines, including keratinocyte-derived chemokine, IL-6, and IL-1 beta. Similarly, both the accumulation of neutrophils in the lung as well as enhanced lung permeability were reduced when anti-HMGB1 antibodies were injected after hemorrhage. These results demonstrate that hemorrhage results in increased HMGB1 expression in the lungs, primarily through neutrophil sources, and that HMGB1 participates in hemorrhage-induced acute lung injury.
[show abstract][hide abstract] ABSTRACT: This study was performed to examine the putative role of high mobility group box (HMGB) protein in the pathogenesis of acute lung injury (ALI). Observations were made (1) in 21 patients who were septic with ALI and 15 patients with normal lung function and (2) in a mouse model 24 hours after intratracheal instillation of lipopolysaccharide (LPS). The concentrations of HMGB1 were increased in plasma and lung epithelial lining fluid of patients with ALI and mice instilled with LPS. LPS-induced ALI was mitigated by anti-HMGB1 antibody. Although this protein was not detected in the plasma of control humans or mice, the concentrations of HMGB1 in lung epithelial lining fluid or in bronchoalveolar lavage fluid were unexpectedly high. The nuclear expression of HMGB1 was apparent in epithelial cells surrounding terminal bronchioles in normal mice, whereas its nuclear and cytoplasmic expression was observed in alveolar macrophages in LPS-instilled mice. Lung instillation of HMGB2 did not cause as much inflammation as HMGB1. Extracellular HMGB1 may play a key role in the pathogenesis of clinical and experimental ALI. However, its expression in normal airways is noteworthy and suggests that it also plays a physiologic role in the lung.
American Journal of Respiratory and Critical Care Medicine 01/2005; 170(12):1310-6. · 11.04 Impact Factor
[show abstract][hide abstract] ABSTRACT: High mobility group box chromosomal protein 1 (HMGB-1), a nuclear DNA binding protein, was recently rediscovered as a new proinflammatory cytokine. The purpose of this study was to demonstrate HMGB-1 expression in vivo and to identify the role of HMGB-1 in the pathogenesis of rheumatoid arthritis (RA).
HMGB-1 concentrations in synovial fluid (SF) and serum from RA and osteoarthritis (OA) patients were measured by immunoblot analysis. The protein's specific receptor, receptor for advanced glycation end products (RAGE), was examined in SF macrophages (SFMs). We measured levels of proinflammatory cytokines released by SFMs treated with HMGB-1 via enzyme-linked immunosorbent assay and used soluble RAGE (sRAGE) to block the release of tumor necrosis factor alpha (TNFalpha). Immunohistochemical analysis and immunofluorescence assay were employed to examine localization of HMGB-1 in RA synovium and its translocation in SFMs after TNFalpha stimulation.
HMGB-1 concentrations were significantly higher in SF of RA patients than in that of OA patients. SFMs expressed RAGE and released TNFalpha, interleukin-1beta (IL-1beta), and IL-6 upon stimulation with HMGB-1. HMGB-1 was found in CD68-positive cells of RA synovium, and TNFalpha stimulation translocated HMGB-1 from the nucleus to the cytosol in SFMs. Blockade by sRAGE inhibited the release of TNFalpha from SFMs.
HMGB-1 was more strongly expressed in SF of RA patients than in that of OA patients, inducing the release of proinflammatory cytokines from SFMs. HMGB-1 plays a pivotal role in the pathogenesis of RA and may be an original target of therapy as a novel cytokine.
[show abstract][hide abstract] ABSTRACT: Although oxidized lipoproteins may play an important role in the progression of atherosclerosis, no report has mentioned the significance of oxidized lipoprotein (a) (Lp[a]) in the pathogenesis of cardiovascular disease. Initially, we compared the mitogenic actions of Lp(a) and oxidized Lp(a) on human vascular smooth muscle cells (VSMC). Lp(a) significantly stimulated the growth of human VSMC in a dose-dependent manner, whereas oxidized Lp(a) showed a stronger stimulatory action on VSMC growth than native Lp(a). Interestingly, antioxidants probucol and fluvastatin inhibited the oxidation of Lp(a). Moreover, the stimulatory effect of oxidized Lp(a) on human VSMC growth was significantly inhibited by probucol. Finally, we elucidated the molecular mechanisms of how Lp(a) stimulated the growth of VSMC. Extracellular signal-regulated kinase (ERK), as those controlled by kinases, modulate critical cellular functions such as cell growth, differentiation, and apoptosis, was transiently phosphorylated by oxidized Lp(a) as well as native Lp(a) from 5 minutes, and the phosphorylation disappeared within 30 minutes. The degree of ERK phosphorylation by oxidized Lp(a) was much higher than that by native Lp(a). Administration of a specific inhibitor of MEK, PD 98059, significantly attenuated VSMC growth induced by native Lp(a) or oxidized Lp(a) in a dose-dependent manner (P<0.01). The current study demonstrated that oxidized Lp(a) is more potent than native Lp(a) in stimulating VSMC growth. Oxidized Lp(a) may play an important role in the pathogenesis of vascular disease.