JH-4 reduces HMGB1-mediated septic responses and improves survival rate in septic mice

Inhibition of high mobility group box 1 (HMGB1) and restoration of endothelial integrity are emerging as attractive therapeutic strategies for the management of severe vascular inflammatory diseases. Recently, we found that JH-4, a synthesized decursin derivative, exhibited a strong anti-Hutchinson-Gilford progeria syndrome by efficiently blocking progerin-lamin A/C binding. In this study, we examined the effects of JH-4 on HMGB1-mediated septic responses and the survival rate in a mouse sepsis model. The anti-inflammatory activities of JH-4 were monitored based on its effects on lipopolysaccharide- or cecal ligation and puncture (CLP)-mediated release of HMGB1. The antiseptic activities of JH-4 were determined by measuring permeability, leukocyte adhesion, migration, and the activation of proinflammatory proteins in HMGB1-activated human umbilical vein endothelial cells and mice. JH-4 inhibited the release of HMGB1 and downregulated HMGB1-dependent inflammatory responses in human endothelial cells. JH-4 also inhibited HMGB1-mediated hyperpermeability and leukocyte migration in mice. In addition, treatment with JH-4 reduced CLP-induced release of HMGB1, sepsis-related mortality, and pulmonary injury in vivo. Our results indicate that JH-4 is a possible therapeutic agent to treat various severe vascular inflammatory diseases via the inhibition of the HMGB1 signaling pathway.     

Inhibitory functions of maslinic acid,a natural triterpene,on HMGB1-mediated septic responses

BackgroundMaslinic acid (MA), a natural triterpenoid from Olea europaea, prevents oxidative stress and pro-inflammatory cytokine generation. High mobility group box 1 (HMGB1) has been recognized as a late mediator of sepsis, and the inhibition of the release of HMGB1 and the recovery of vascular barrier integrity have emerged as attractive therapeutic strategies for the management of sepsis.MethodsWe tested the hypothesis that MA induces sirtuin 1 and heme oxygenase-1, which inhibit the release of HMGB1 in lipopolysaccharide (LPS)-stimulated cells, thus inhibiting HMGB1-induced hyperpermeability and increasing the survival of septic mice. MA was administered after LPS or HMGB1 challenge, and the antiseptic activity of MA was determined based on permeability, the activation of pro-inflammatory proteins, and the production of markers for tissue injury in HMGB1-activated human umbilical vein endothelial cells (HUVECs) and a cecal ligation and puncture (CLP)-induced sepsis mouse model.ResultsMA significantly reduced the release of HMGB1 in LPS-activated HUVECs and attenuated the CLP-induced release of HMGB1. Additionally, MA alleviated HMGB1-mediated vascular disruption and inhibited hyperpermeability in mice, and in vivo analysis revealed that MA reduced sepsis-related mortality and tissue injury.ConclusionTaken together, the present results suggest that MA reduced HMGB1 release and septic mortality and thus may be useful in the treatment of sepsis.     

Association of high mobility group box-1 protein levels with sepsis and outcome of severely burned patients

BackgroundThe study was performed to observe the systemic release and kinetics of high mobility group box-1 protein (HMGB1) in burned patients.Methods106 patients were included, and they were divided into three groups with different burn sizes: group I, group II and group III. Healthy volunteers served as normal controls (n = 25). The peripheral blood samples were collected on postburn days (PBD) 1, 3, 7, 14, and 21. The blood samples were used to detect levels of HMGB1 in plasma by ELISA kits for human. Gene expression of HMGB1 in peripheral blood mononuclear cells was assessed by real-time quantitative PCR taking GAPDH as the internal standard.ResultsThe levels of HMGB1 were significantly elevated on PBD 1–21 in patients with various burn sizes compared with normal controls, and there were obvious differences between group I and group III. The HMGB1 levels were significantly higher in septic patients than those without sepsis on PBD 7–21. Among septic patients, the HMGB1 levels in the survival group were markedly lower than those with fatal outcome on PBD 3–21.ConclusionsExtensive burn injury could result in significantly increased HMGB1 levels, which appears to be associated with the development of sepsis and fatal outcome of major burns.     

Anti‐inflammatory activities of isorhamnetin‐3‐O‐galactoside against HMGB1‐induced inflammatory responses in both HUVECs and CLP‐induced septic mice

High mobility group box 1 (HMGB1) protein is a crucial nuclear cytokine that elicits severe vascular inflammatory diseases. Oenanthe javanica (water dropwort) extract has anti‐arrhythmic, neuroprotective and anti‐diabetic activity. However, isorhamnetin‐3‐O‐galactoside (I3G), an active compound from O. javanica, is not researched well for its biological activity. Here, we investigated the anti‐inflammatory activities of I3G by monitoring the effects of I3G on the lipopolysaccharide (LPS) or cecal ligation and puncture (CLP)‐mediated release of HMGB1 and HMGB1 or CLP‐mediated modulation of inflammatory responses. I3G potently inhibited the release of HMGB1 and down‐regulated HMGB1‐dependent inflammatory responses in human endothelial cells. I3G also inhibited HMGB1‐mediated hyperpermeability and leukocyte migration in mice. Further studies revealed that I3G suppressed the production of tumor necrosis factor‐α and activation of nuclear factor‐κB by HMGB1. In addition, I3G reduced CLP‐induced HMGB1 release and sepsis‐related mortality. Given these results, I3G should be viewed as a candidate therapeutic agent for the treatment of severe vascular inflammatory diseases such as sepsis or septic shock via inhibition of the HMGB1 signaling pathway. J. Cell. Biochem. 114: 336–345, 2013. © 2012 Wiley Periodicals, Inc.     

Splenectomy protects against sepsis lethality and reduces serum HMGB1 levels

High mobility group box 1 (HMGB1) is a critical mediator of lethal sepsis. Previously, we showed that apoptotic cells can activate macrophages to release HMGB1. During sepsis, apoptosis occurs primarily in lymphoid organs, including the spleen and thymus. Currently, it is unclear whether this accelerated lymphoid organ apoptosis contributes to systemic release of HMGB1 in sepsis. In this study, we report that splenectomy significantly reduces systemic HMGB1 release and improves survival in mice with polymicrobial sepsis. Treatment with a broad-spectrum caspase inhibitor reduces systemic lymphocyte apoptosis, suppresses circulating HMGB1 concentrations, and improves survival during polymicrobial sepsis, but fails to protect septic mice following splenectomy. These findings indicate that apoptosis in the spleen is essential to the pathogenesis of HMGB1-mediated sepsis lethality.     

Persicarin is anti‐inflammatory mediator against HMGB1‐induced inflammatory responses in HUVECs and in CLP‐induced sepsis mice

High mobility group box 1 (HMGB1) protein is a crucial nuclear cytokine that mediates inflammatory responses, whereas persicarin is an active compound from Oenanthe javanica that has been widely researched for its neuroprotective and antioxidant activities. However, little is known of the effects of persicarin on HMGB1‐mediated inflammatory response. Here, we investigated this issue by monitoring the effects of persicarin on the lipopolysaccharide (LPS) and on the cecal ligation and puncture (CLP)‐mediated releases of HMGB1 and the effects of persicarin on the HMGB1‐mediated modulation of inflammatory response. Persicarin potently inhibited the release of HMGB1 and down‐regulated HMGB1‐dependent inflammatory responses in human endothelial cells, and inhibited HMGB1‐mediated hyperpermeability and leukocyte migration in mice. Furthermore, persicarin reduced CLP‐induced HMGB1 release and sepsis‐related mortality. Given these results, persicarin should be viewed as a candidate therapeutic for the treatment of severe vascular inflammatory diseases, such as, sepsis or septic shock. J. Cell. Physiol. 228: 696–703, 2013. © 2012 Wiley Periodicals, Inc.     

Enhanced dendritic cell survival attenuates lipopolysaccharide-induced immunosuppression and increases resistance to lethal endotoxic shock

Impaired immune function and associated immunosuppression are hallmarks of septic syndromes. As part of an overall deactivation of the immune system, profound depletion of dendritic cells (DCs) occurs in both septic patients and septic mice. Such depletion of DCs is potentially associated with immunosuppression and with failure to induce a protective Th1 immune response; it may equally be predictive of fatal outcome in septic patients. To evaluate the impact of enhanced DC survival on LPS-induced immunosuppression and on survival after LPS-induced septic shock, we created a transgenic mouse model specifically overexpressing the human form of the antiapoptotic protein Bcl-2 in DCs (DC-hBcl-2 mice). DCs derived from DC-hBcl-2 mice exhibited higher resistance to maturation-induced apoptosis after LPS treatment both in vitro and in vivo. Moreover, prolongation of DC survival diminished sublethal LPS-induced DC loss and immunosuppression, with maintenance of the differentiation potential of Th1 cells and enhanced T cell activation. Such modulation of the immune response appears to constitute a key feature of the attenuated mortality observed after LPS-induced shock in DC-hBcl-2 mice. Our study therefore identifies DC death as a key determinant of endotoxin-induced immunosuppression and mortality in mice.     

Glucan phosphate attenuates myocardial HMGB1 translocation in severe sepsis through inhibiting NF-κB activation

Myocardial dysfunction is a major consequence of septic shock and contributes to the high mortality of sepsis. High-mobility group box 1 (HMGB1) serves as a late mediator of lethality in sepsis. We have reported that glucan phosphate (GP) attenuates cardiac dysfunction and increases survival in cecal ligation and puncture (CLP)-induced septic mice. In the present study, we examined the effect of GP on HMGB1 translocation from the nucleus to the cytoplasm in the myocardium of septic mice. GP was administered to mice 1 h before induction of CLP. Sham-operated mice served as control. The levels of HMGB1, Toll-like receptor 4 (TLR4), and NF-κB binding activity were examined. In an in vitro study, H9C2 cardiomyoblasts were treated with lipopolysaccharide (LPS) in the presence or absence of GP. H9C2 cells were also transfected with Ad5-IκBα mutant, a super repressor of NF-κB activity, before LPS stimulation. CLP significantly increased the levels of HMGB1, TLR4, and NF-κB binding activity in the myocardium. In contrast, GP administration attenuated CLP-induced HMGB1 translocation from the nucleus to the cytoplasm and reduced CLP-induced increases in TLR4 and NF-κB activity in the myocardium. In vitro studies showed that GP prevented LPS-induced HMGB1 translocation and NF-κB binding activity. Blocking NF-κB binding activity by Ad5-IκBα attenuated LPS-induced HMGB1 translocation. GP administration also reduced the LPS-stimulated interaction of HMGB1 with TLR4. These data suggest that attenuation of HMGB1 translocation by GP is mediated through inhibition of NF-κB activation in CLP-induced sepsis and that activation of NF-κB is required for HMGB1 translocation.     

Induction of release of tumor necrosis factor and IL-6 from human mononuclear cells by Bacteroides strains

The role of anaerobic Gram-negative bacteria in inducing cytokines during mixed infections involving aerobic and anaerobic bacteria is relatively poorly defined. The purpose of this study was to establish whether or not intact Bacteroides fragilis and related species, isolated from severe infections and from the faeces of healthy persons are capable of releasing tumor necrosis factor (TNF) and IL-6 from human mononuclear cells and whole blood. The purified lipopolysaccharides of Bacteroides fragilis strain (No. 7), extracted by the aqueous phenol method from BHI cultures and from BHI culture supplemented with 5% horse serum, were also tested. TNF release was detected by the WEHI 164-dependent bioassay and IL-6 production by the B-9 cell-dependent bioassay. Heat-inactivated Bacteroides strains belonging to different species were able to induce TNF (1x10(1)-5x10(2) U/mL) and IL-6 (1x10(1)-5x10(5) pg/mL) release from human mononuclear cells. When whole blood was used, the production of TNF and IL-6 was more pronounced (very probably because of the presence of certain serum factors). The culturing conditions (the presence of 5% horse serum in the BHI broth) influenced the inducing activity of almost all strains tested. The isolated lipopolysaccharide of Bacteroides fragilis strain No. 7 proved to have a rough profile on PAGE. There were no differences in TNF and IL-6 induction when the lipopolysaccharides of the strain was cultured in BHI or in BHI supplemented with 5% horse serum. Bacteroides strains often outnumber Enterobacteriaceae in the faeces and in mixed infections, and their role in inducing and/or modulating the host response in septic shock should not be overlooked.     

The Heme Oxygenase-1 Inducer THI-56 Negatively Regulates iNOS Expression and HMGB1 Release in LPS-Activated RAW 264.7 Cells and CLP-Induced Septic Mice

The nuclear DNA binding protein high mobility group box 1 (HMGB1) has recently been suggested to act as a late mediator of septic shock. The effect of ((S)-6,7-dihydroxy-1-(4-hydroxynaphthylmethyl)-1,2,3,4-tetrahydroisoquinoline alkaloid, also known as THI-56, in an experimental model of sepsis was investigated. THI-56 exhibited potent anti-inflammatory properties in response to LPS in RAW 264.7 cells. In particular, THI-56 significantly inhibited the expression of inducible nitric oxide synthase (iNOS) and the release of HMGB1 in activated macrophages. THI-56 activated NE-F2-regulated factor 2 (Nrf-2)/heme oxygenase 1 (HO-1). The specific knockdown of the HO-1 gene by HO-1 siRNA significantly reversed the inhibitory effects of THI-56 on iNOS expression and HMGB1 release in LPS-stimulated macrophages. Importantly, THI-56 administration protected animals from death induced by either a lethal dose of LPS or cecal ligation and puncture (CLP). Furthermore, the ALT, AST, BUN, creatinine, and HMGB1 levels in the blood were significantly increased in CLP-induced septic mice, and the administration of THI-56 reduced these levels in a concentration-dependent and zinc protoporphyrin IX (ZnPPIX)-sensitive manner. In addition, the administration of THI-56 significantly ameliorated not only lung damage but also macrophage infiltration in the livers of CLP-induced septic mice, and these effects were also abrogated in the presence of ZnPPIX. Thus, we conclude that THI-56 significantly attenuates the proinflammatory response induced by LPS and reduces organ damage in a CLP-induced sepsis model through the upregulation of Nrf-2/HO-1.     

TNFalpha release from peripheral blood leukocytes depends on a CRM1-mediated nuclear export

Tumor necrosis factor-alpha (TNFalpha) is a potent pro-inflammatory cytokine that plays a major role in the pathogenesis of acute and chronic inflammatory disorders such as septic shock and arthritis, respectively. Leukocytes stimulated with inflammatory signals such as lipopolysaccharide (LPS) are the predominant producers of TNFalpha, and thus control of TNFalpha release from stimulated leukocytes represents a potential therapeutic target. Here, we report that leptomycin B (LMB), a specific inhibitor of CRM1-dependent nuclear protein export, inhibits TNFalpha release from LPS-stimulated human peripheral blood neutrophils and mononuclear cells. In addition, immunofluorescence confocal microscopy and immunoblotting analysis indicate that TNFalpha is localized in the nucleus of human neutrophils and mononuclear cells. This study demonstrates that the cellular release of TNFalpha from stimulated leukocytes is mediated by the CRM1-dependent nuclear export mechanism. Inhibition of CRM1-dependent cellular release of TNFalpha could thus provide a novel therapeutic approach for disorders involving excessive TNFalpha release.     

Anti-HMGB1 neutralizing antibody ameliorates gut barrier dysfunction and improves survival after hemorrhagic shock

Intestinal barrier dysfunction occurs following hemorrhagic shock and resuscitation (HS/R). High-mobility group B1 (HMGB1) has been shown to increase the permeability of Caco-2 human enterocyte-like epithelial monolayers in vitro. In this study, we found that serum concentrations of HMGB1 were higher in blood samples obtained from 25 trauma victims with hemorrhagic shock than in 9 normal volunteers. We also studied whether treatment with anti-HMGB1 antibody can ameliorate HS/R-induced gut barrier dysfunction in mice. Animals were shocked by withdrawal of blood to maintain mean arterial pressure at 25 to 30 mmHg for 2 h. After resuscitation with shed blood plus Ringer's lactate solution, the mice were treated with either anti-HMGB1 antibody or nonimmune rabbit IgG. Serum HMGB1 concentrations were significantly higher in trauma victims than control mice. Treatment with anti-HMGB1 antibody improved survival at 24 h and ameliorated the development of ileal mucosal hyperpermeability to FITC-labeled dextran. At 24 h after HS/R, treatment with anti-HMGB1 antibody decreased bacterial translocation to mesenteric lymph nodes and was associated with lower circulating concentrations of IL-6 and IL-10. These data support the notion that HMGB1 is a mediator of HS/R-induced gut barrier dysfunction and suggest that anti-HMGB1 antibodies warrant further evaluation as a therapeutic to ameliorate the morbidity of HS/R in trauma patients.     

NK but not CD1-restricted NKT cells facilitate systemic inflammation during polymicrobial intra-abdominal sepsis

Evidence suggests that NK and NKT cells contribute to inflammation and mortality during septic shock caused by cecal ligation and puncture (CLP). However, the specific contributions of these cell types to the pathogenesis of CLP-induced septic shock have not been fully defined. The goal of the present study was to determine the mechanisms by which NK and NKT cells mediate the host response to CLP. Control, NK cell-deficient, and NKT cell-deficient mice underwent CLP. Survival, cytokine production, and bacterial clearance were measured. NK cell trafficking and interaction with myeloid cells was also studied. Results show that mice treated with anti-asialoGM1 (NK cell deficient) or anti-NK1.1 (NK/NKT cell deficient) show less systemic inflammation and have improved survival compared with IgG-treated controls. CD1 knockout mice (NKT cell deficient) did not demonstrate decreased cytokine production or improved survival compared with wild type mice. Trafficking studies show migration of NK cells from blood and spleen into the inflamed peritoneal cavity where they appear to facilitate the activation of peritoneal macrophages (F4-80(+)GR-1(-)) and F4-80(+)Gr-1(+) myeloid cells. These findings indicate that NK but not CD1-restricted NKT cells contribute to acute CLP-induced inflammation. NK cells appear to mediate their proinflammatory functions during septic shock, in part, by migration into the peritoneal cavity and amplification of the proinflammatory activities of specific myeloid cell populations. These findings provide new insights into the mechanisms used by NK cells to facilitate acute inflammation during septic shock.     

Dengue Virus Infection Mediates HMGB1 Release from Monocytes Involving PCAF Acetylase Complex and Induces Vascular Leakage in Endothelial Cells

High mobility group box 1 (HMGB1) protein is released from cells as a pro-inflammatory cytokine in response to an injury or infection. During dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS), a number of pro-inflammatory cytokines are released, contributing to disease pathogenesis. In this study, the release of HMGB1 from human myelogenous leukemia cell line K562 and primary peripheral blood monocytes (PBM) cells was examined during dengue virus (DV)-infection. HMGB1 was shown to translocate from cell nuclei to the cytoplasm in both K562- and PBM-infected cells. The translocation of HMGB1 from the nucleus to the cytoplasm was shown to be mediated by the host cell p300/CBP-associated factor (PCAF) acetylase complex in K562 cells. In addition, DV capsid protein was observed to be the putative viral protein in actuating HMGB1 migration from the nucleus to cytoplasm through the involvement of PCAF acetylase. HMGB1 was released from DV-infected K562 cells into the extracellular milieu in a multiplicity of infection (M.O.I.)-independent manner and its release can be inhibited by the addition of 1-5 mM of ethyl pyruvate (EP) in a dose-dependent manner. Application of DV-infected K562 cell culture supernatants to primary endothelial cells induced vascular permeability. In contrast, supernatants from DV-infected K562 cells treated with EP or HMGB1 neutralizing antibody were observed to maintain the structural integrity of the vascular barrier.     

Killing of Escherichia coli by mononuclear phagocytes and neutrophils stimulated in vitro with beta-1,3-D-polyglucose derivatives.

Human monocytes, human peritoneal macrophages, mouse peritoneal macrophages and human peripheral neutrophils pretreated with beta-1,3-D-polyglucose derivatives showed pronounced bactericidal capacity to Escherichia coli compared to control cells. The increased bactericidal capacity was detectable in mononuclear phagocytes over a wide range of concentrations of bacteria. Granulocytes, however, showed bactericidal capacity only at low concentrations of bacteria. The pretreated mononuclear phagocytes released significant amounts of IL-1 and PGE2. However, there was no significant release of tumor necrosis factor (TNF). By incubating unstimulated cells with purified IL-1 and TNF, the bactericidal activity of neutrophils and mononuclear phagocytes was enhanced. Our data indicate that the inability of neutrophils stimulated with beta-1,3-D-polyglucose derivatives to kill large numbers of bacteria could be overcome by a combined treatment with purified IL-1 or TNF in addition to beta-1,3-D-polyglucose derivatives. By incubating unstimulated cells with medium from beta-1,3-D-polyglucose-treated human peritoneal macrophages, the bactericidal activity of the cells was enhanced to the same extent as cells pretreated with purified TNF and IL-1. Cells incubated with IL-1-depleted medium from beta-1,3-D-polyglucose-treated human peritoneal macrophages, showed reduced bactericidal activity compared to cells incubated with undepleted medium. These studies demonstrate that beta-1,3-D-polyglucose-treated mononuclear phagocytes and neutrophils show enhanced bactericidal activity. The enhanced activity is partly caused by stimulation of the cells with IL-1 released from mononuclear phagocytes and partly by other unknown effects of beta-1,3-D-polyglucose derivatives on both mononuclear phagocytes and neutrophils.     

Protease-Activated Receptor 4 Induces Bladder Pain through High Mobility Group Box-1

Pain is the significant presenting symptom in Interstitial Cystitis/Painful Bladder Syndrome (IC/PBS). Activation of urothelial protease activated receptor 4 (PAR4) causes pain through release of urothelial macrophage migration inhibitory factor (MIF). High Mobility Group Box-1 (HMGB1), a chromatin-binding protein, mediates bladder pain (but not inflammation) in an experimental model (cyclophosphamide) of cystitis. To determine if PAR4-induced bladder hypersensitivity depends on HMGB1 downstream, we tested whether: 1) bladder PAR4 stimulation affected urothelial HMGB1 release; 2) blocking MIF inhibited urothelial HMGB1 release; and 3) blocking HMGB1 prevented PAR4-induced bladder hypersensitivity. HMGB1 release was examined in immortalized human urothelial cultures (UROtsa) exposed to PAR4-activating peptide (PAR4-AP; 100 μM; 2 hours) or scrambled control peptide. Female C57BL/6 mice, pretreated with a HMGB1 inhibitor (glycyrrhizin: 50 mg/kg; ip) or vehicle, received intravesical PAR4-AP or a control peptide (100 μM; 1 hour) to determine 1) HMGB1 levels at 1 hour in the intravesical fluid (released HMGB1) and urothelium, and 2) abdominal hypersensitivity to von Frey filament stimulation 24 hours later. We also tested mice pretreated with a MIF blocker (ISO-1: 20 mg/kg; ip) to determine whether MIF mediated PAR4-induced urothelial HMGB1 release. PAR4-AP triggered HMGB1 release from human (in vitro) and mice (in vivo) urothelial cells. Intravesical PAR4 activation elicited abdominal hypersensitivity in mice that was prevented by blocking HMGB1. MIF inhibition prevented PAR4-mediated HMGB1 release from mouse urothelium. Urothelial MIF and HGMB1 represent novel targets for therapeutic intervention in bladder pain conditions.     

人HMGB1酸性尾端对其抗菌活性的影响

为研究人高迁移率族蛋白B1(high-mobility group box-1HMGB1)酸性尾端对其抗菌活 性的影响,提取人外周血单个核细胞总RNA,经RT\|PCR扩增得到编码人HMGB1的cDNA及其缺失酸性尾端的突变体cDNA(mcDNA),原核表达载体pQE\|80L分别表达重组人HMGB1蛋白(rhHMGB1)及缺失酸性尾端的突变体蛋白(mrhHMGB1),经Ni^2+_- NTA亲和层析柱纯化两种蛋白.通过试管稀释法、琼脂扩散法两种体外抗菌实验观察,并比较rhHMGB1mrhHMGB1抗菌活性的差异.实验结果显示,rhHMGB1对大肠杆菌JM109、ATCC2592 2、DH5α有明确的抗菌活性,其抗菌活性强弱依次为JM109>ATCC25922>DH5α,而mrhHMGB1 对大肠杆菌JM109、ATCC25922、DH5α则均无抗菌活性.实验结果表明,人HMGB1的酸性尾端对其抗菌活性的发挥至关重要,此研究为进一步探讨人HMGB1抗菌功能的机制奠定了基础.     

TNF-α induced secretion of HMGB1 from non-immune canine mammary epithelial cells (MTH53A)

BackgroundMammary neoplasias are one of the most frequent and spontaneously occurring malignancies in dogs and humans. Due to the similar anatomy of the mammary gland in both species, the dog has become an important animal model for this cancer entity. In human breast carcinomas, the overexpression of a protein named high-mobility group box 1 (HMGB1) was reported. Cells of the immune system were described to release HMGB1 actively exerting cytokine function. Thereby it is involved in the immune system activation, tissue repair, and cell migration. Passive release of HMGB1 by necrotic cells at sites of tissue damage or in necrotic hypoxic regions of tumors induces cellular responses e.g. release of proinflammatory cytokines leading to elevated inflammatory response and neo-vascularization of necrotic tumor areas.Herein we investigated if a time-dependent stimulation with the separately applied proinflammatory cytokines TNF-α and IFN-γ can cause secretion of HMGB1 in a non-immune related HMGB1-non-secreting epithelial canine mammary cell line (MTH53A) derived from non-neoplastic tissue.MethodsThe canine cell line was transfected with recombinant HMGB1 bicistronic expression vectors and stimulated after transfection with the respective cytokine independently for 6, 24 and 48 h. HMGB1 protein detection was performed by Western blot analysis and quantified a by enzyme-linked immunosorbent assay. Live cell laser scanning multiphoton microscopy of MTH53A cells expressing a HMGB1–GFP fusion protein was performed in order to examine, if secretion of HMGB1 under cytokine stimulating conditions is also visible by fluorescence imaging.ResultsThe observed HMGB1 release kinetics showed a clearly time-dependent manner with a peak release 24 h after TNF-α stimulation, while stimulation with IFN-γ had only small effects on the HMGB1 release. Multiphoton HMGB1 live cell microscopy showed diffuse cell membrane structure changes 29 h after cytokine-stimulation but no clear secretion of HMGB1–GFP after TNF-α stimulation was visible.ConclusionOur results demonstrate that non-immune HMGB1-non-secreting cells of epithelial origin derived from mammary non-neoplastic tissue can be induced to release HMGB1 by single cytokine application. This indicates that tumor and surrounding tissue can be stimulated by tumor present inflammatory and necrotic cytokines to release HMGB1 acting as neo-vascularizing factor thus promoting tumor growth.     

Superantigen staphylococcal enterotoxin C1 inhibits the growth of bladder cancer

Superantigens can induce cell-mediated cytotoxicity preferentially against MHC II-positive target cells with large amounts of inflammatory cytokines releasing. In this study, superantigen staphylococcal enterotoxin C (SEC) 1 was investigated to evaluate its potential in bladder cancer immunotherapy in vitro and in vivo. Our results revealed that SEC1 could stimulate the proliferation of human peripheral blood mononuclear cells (PBMCs) in a dose-dependent manner, accompanied with the release of interleukin-2, interferon-γ, and tumor necrosis factor-α, and increased the population of CD4⁺ T cells and CD8⁺ T cells. PBMCs stimulated by SEC1 could initiate significant cytotoxicity towards human bladder cancer cells in vitro. The results of in vivo antitumor experiment indicated that SEC1 could decrease the rate of tumor formation and prolong the survival time of tumor-bearing mice. Our study demonstrated that SEC1 inhibited the growth of bladder cancer. And it is also suggested that SEC1 may become a candidate for bladder cancer immunotherapy.     

Cholinergic agonists inhibit HMGB1 release and improve survival in experimental sepsis

Physiological anti-inflammatory mechanisms can potentially be exploited for the treatment of inflammatory disorders. Here we report that the neurotransmitter acetylcholine inhibits HMGB1 release from human macrophages by signaling through a nicotinic acetylcholine receptor. Nicotine, a selective cholinergic agonist, is more efficient than acetylcholine and inhibits HMGB1 release induced by either endotoxin or tumor necrosis factor-alpha (TNF-alpha). Nicotinic stimulation prevents activation of the NF-kappaB pathway and inhibits HMGB1 secretion through a specific 'nicotinic anti-inflammatory pathway' that requires the alpha7 nicotinic acetylcholine receptor (alpha7nAChR). In vivo, treatment with nicotine attenuates serum HMGB1 levels and improves survival in experimental models of sepsis, even when treatment is started after the onset of the disease. These results reveal acetylcholine as the first known physiological inhibitor of HMGB1 release from human macrophages and suggest that selective nicotinic agonists for the alpha7nAChR might have therapeutic potential for the treatment of sepsis.