Urine levels of HMGB1 in Systemic Lupus Erythematosus patients with and without renal manifestations

Introduction

Lupus nephritis (LN) is a severe and frequent manifestation of systemic lupus erythematosus (SLE). Its pathogenesis has not been fully elucidated but immune complexes are considered to contribute to the inflammatory pathology in LN. High Mobility Group Box 1 (HMGB1) is a nuclear non-histone protein which is secreted from different types of cells during activation and/or cell death and may act as a pro-inflammatory mediator, alone or as part of DNA-containing immune complexes in SLE. Urinary excretion of HMGB1 might reflect renal inflammatory injury. To assess whether urinary HMGB1 reflects renal inflammation we determined serum levels of HMGB1 simultaneously with its urinary levels in SLE patients with and without LN in comparison to healthy controls (HC). We also analyzed urinary HMGB1 levels in relation with clinical and serological disease activity.

Methods

The study population consisted of 69 SLE patients and 17 HC. Twenty-one patients had biopsy proven active LN, 15 patients had a history of LN without current activity, and 33 patients had non-renal SLE. Serum and urine levels of HMGB1 were both measured by western blotting. Clinical and serological parameters were assessed according to routine procedures. In 17 patients with active LN a parallel analysis was performed on the expression of HMGB1 in renal biopsies.

Results

Serum and urinary levels of HMGB1 were significantly increased in patients with active LN compared to patients without active LN and HC. Similarly, renal tissue of active LN patients showed strong expression of HMGB1 at cytoplasmic and extracellular sites suggesting active release of HMGB1. Serum and urinary levels in patients without active LN were also significantly higher compared to HC. Urinary HMGB1 levels correlated with SLEDAI, and showed a negative correlation with complement C3 and C4.

Conclusion

Levels of HMGB1 in urine of SLE patients, in particular in those with active LN, are increased and correlate with SLEDAI scores. Renal tissue of LN patients shows increased release of nuclear HMGB1 compared to control renal tissue. HMGB1, although at lower levels, is, however, also present in the urine of patients without active LN. These data suggest that urinary HMGB1 might reflect both local renal inflammation as well as systemic inflammation.     

Is serum HMGB1 a biomarker in ANCA-associated vasculitis?

Background

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) are systemic inflammatory disorders that include granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), Churg-Strauss syndrome and renal limited vasculitis (RLV). Extracellular high-mobility group box 1 (HMGB1) acts as an alarmin and has been shown to be a biomarker of disease activity as well as an autoantigen in systemic lupus erythematosus (SLE) and, possibly, in AAV. This study aims to assess antibodies against HMGB1 and HMGB1 levels as biomarkers for AAV disease activity and predictors of relapsing disease.

Methods

AAV patients with active disease and healthy controls (HC) were evaluated for anti-HMGB1 antibodies while serum HMGB1 levels were measured longitudinally in AAV patients at presentation, during remission, prior to and at relapses.

Results

HMGB1 levels were similar between AAV patients at presentation (n = 52) and HC (n = 35) (2.64 ± 1.80 ng/ml vs. 2.39 ± 1.09 ng/ml; P = 0.422) and no difference regarding HMGB1 levels could be found among AAV disease subsets (GPA: 2.66 ± 1.83 ng/ml vs. MPA: 3.11 ± 1.91 ng/ml vs. RLV: 1.92 ± 1.48 ng/ml; P = 0.369). AAV patients with renal involvement had lower HMGB1 levels than patients without renal involvement at presentation (2.35 ± 1.48 ng/ml vs. 3.52 ± 2.41 ng/ml; P = 0.042). A negative correlation was observed between HMGB1 levels and 24-hour proteinuria (ρ = -0.361, P = 0.028). Forty-nine AAV patients were evaluated for HMGB1 levels during follow-up and no differences were observed between relapsing and nonrelapsing patients (P = 0.350). No significant increase in HMGB1 levels was observed prior to a relapse compared with the remission period and changes in HMGB1 levels were not associated with an increased risk for relapse in AAV. Positivity for anti-HMGB1 antibodies was low in patients with active AAV (three out of 24 patients).

Conclusions

Serum HMGB1 levels at presentation are not increased and are lower in patients with renal involvement. Relapses are not preceded or accompanied by significant rises in HMGB1 levels and changes in HMGB1 levels are not related to ensuing relapses. Anti-HMGB1 antibodies are present in only a few patients in AAV. In contrast to SLE, HMGB1 is not a useful biomarker in AAV.     

HMGB1 Promotes the Development of Pulmonary Arterial Hypertension in Rats

Rationale

Pulmonary arterial hypertension (PAH) is characterized by increased pulmonary vascular resistance leading to right ventricular failure and death. Recent studies have suggested that chronic inflammatory processes are involved in the pathogenesis of PAH. However, the molecular and cellular mechanisms driving inflammation have not been fully elucidated.

Objectives

To elucidate the roles of high mobility group box 1 protein (HMGB1), a ubiquitous DNA-binding protein with extracellular pro-inflammatory activity, in a rat model of PAH.

Methods

Male Sprague-Dawley rats were administered monocrotaline (MCT). Concentrations of HMGB1 in bronchoalveolar lavage fluid (BALF) and serum, and localization of HMGB1 in the lung were examined over time. The protective effects of anti-HMGB1 neutralizing antibody against MCT-induced PAH were tested.

Results

HMGB1 levels in BALF were elevated 1 week after MCT injection, and this elevation preceded increases of other pro-inflammatory cytokines, such as TNF-α, and the development of PAH. In contrast, serum HMGB1 levels were elevated 4 weeks after MCT injection, at which time the rats began to die. Immunohistochemical analyses indicated that HMGB1 was translocated to the extranuclear space in periarterial infiltrating cells, alveolar macrophages, and bronchial epithelial cells of MCT-injected rats. Anti-HMGB1 neutralizing antibody protected rats against MCT-induced lung inflammation, thickening of the pulmonary artery wall, and elevation of right ventricular systolic pressure, and significantly improved the survival of the MCT-induced PAH rats.

Conclusions

Our results identify extracellular HMGB1 as a promoting factor for MCT-induced PAH. The blockade of HMGB1 activity improved survival of MCT-induced PAH rats, and thus might be a promising therapy for the treatment of PAH.     

HMGB1 is secreted by immunostimulated enterocytes and contributes to cytomix-induced hyperpermeability of Caco-2 monolayers

High-mobility group box 1 (HMGB1), a cytokine-like proinflammatory protein, is secreted by activated macrophages and released by necrotic cells. We hypothesized that immunostimulated enterocytes might be another source for this mediator. Accordingly, Caco-2 cells or primary mouse intestinal epithelial cells (IECs) were incubated with "cytomix" (a mixture of TNF, IL-1, and IFN-) for various periods. HMGB1 in cell culture supernatants was detected by Western blot analysis and visualized in Caco-2 cells with the use of fluorescence confocal and immunotransmission electron microscopy. Caco-2 cells growing on filters in diffusion chambers were stimulated with cytomix for 48 h in the absence or presence of anti-HMGB1 antibody, and permeability to fluorescein isothiocyanate-dextran (average molecular mass, 4 kDa; FD4) was assessed. Cytomix-stimulated Caco-2 cells secreted HMGB1 into the apical but not the basolateral compartments of diffusion chambers. Although undetectable at 6 and 12 h after the start of incubation with cytomix, HMGB1 was present in supernatants after 24 h of incubation. HMGB1 secretion by Caco-2 monolayers also was induced when the cells were exposed to FSL-1, a Toll-like receptor (Tlr)-2 agonist, or flagellin, a Tlr5 agonist, but not lipopolysaccharide, a Tlr4 agonist. Cytomix also induced HMGB1 secretion by primary IECs. Cytoplasmic HMGB1 is localized within vesicles in Caco-2 cells and is secreted, at least in part, associated with exosomes. Incubating Caco-2 cells with cytomix increased FD4 permeation, but this effect was significantly decreased in the presence of anti-HMGB1 antibody. Collectively, these data support the view that HMGB1 is secreted by immunostimulated enterocytes. This process may exacerbate inflammation-induced epithelial hyperpermeability via an autocrine feedback loop. exosome; toll-like receptor; flagellin     

HMGB1 Mediates Endogenous TLR2 Activation and Brain Tumor Regression

Background

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor that carries a 5-y survival rate of 5%. Attempts at eliciting a clinically relevant anti-GBM immune response in brain tumor patients have met with limited success, which is due to brain immune privilege, tumor immune evasion, and a paucity of dendritic cells (DCs) within the central nervous system. Herein we uncovered a novel pathway for the activation of an effective anti-GBM immune response mediated by high-mobility-group box 1 (HMGB1), an alarmin protein released from dying tumor cells, which acts as an endogenous ligand for Toll-like receptor 2 (TLR2) signaling on bone marrow-derived GBM-infiltrating DCs.

Methods and Findings

Using a combined immunotherapy/conditional cytotoxic approach that utilizes adenoviral vectors (Ad) expressing Fms-like tyrosine kinase 3 ligand (Flt3L) and thymidine kinase (TK) delivered into the tumor mass, we demonstrated that CD4⁺ and CD8⁺ T cells were required for tumor regression and immunological memory. Increased numbers of bone marrow-derived, tumor-infiltrating myeloid DCs (mDCs) were observed in response to the therapy. Infiltration of mDCs into the GBM, clonal expansion of antitumor T cells, and induction of an effective anti-GBM immune response were TLR2 dependent. We then proceeded to identify the endogenous ligand responsible for TLR2 signaling on tumor-infiltrating mDCs. We demonstrated that HMGB1 was released from dying tumor cells, in response to Ad-TK (+ gancyclovir [GCV]) treatment. Increased levels of HMGB1 were also detected in the serum of tumor-bearing Ad-Flt3L/Ad-TK (+GCV)-treated mice. Specific activation of TLR2 signaling was induced by supernatants from Ad-TK (+GCV)-treated GBM cells; this activation was blocked by glycyrrhizin (a specific HMGB1 inhibitor) or with antibodies to HMGB1. HMGB1 was also released from melanoma, small cell lung carcinoma, and glioma cells treated with radiation or temozolomide. Administration of either glycyrrhizin or anti-HMGB1 immunoglobulins to tumor-bearing Ad-Flt3L and Ad-TK treated mice, abolished therapeutic efficacy, highlighting the critical role played by HMGB1-mediated TLR2 signaling to elicit tumor regression. Therapeutic efficacy of Ad-Flt3L and Ad-TK (+GCV) treatment was demonstrated in a second glioma model and in an intracranial melanoma model with concomitant increases in the levels of circulating HMGB1.

Conclusions

Our data provide evidence for the molecular and cellular mechanisms that support the rationale for the clinical implementation of antibrain cancer immunotherapies in combination with tumor killing approaches in order to elicit effective antitumor immune responses, and thus, will impact clinical neuro-oncology practice.     

Renal expression and serum levels of high mobility group box 1 protein in lupus nephritis

Introduction

High mobility group box 1 protein (HMGB1) is a nuclear DNA binding protein acting as a pro-inflammatory mediator following extracellular release. HMGB1 has been increasingly recognized as a pathogenic mediator in several inflammatory diseases. Elevated serum levels of HMGB1 have been detected in autoimmune diseases including Systemic lupus erythematosus (SLE). However, the local expression of HMGB1 in active lupus nephritis (LN) is not known. Here we aimed to study both tissue expression and serum levels of HMGB1 in LN patients with active disease and after induction therapy.

Methods

Thirty-five patients with active LN were included. Renal biopsies were performed at baseline and after standard induction therapy; corticosteroids combined with immunosuppressive drugs. The biopsies were evaluated according to the World Health Organization (WHO) classification and renal disease activity was estimated using the British Isles lupus assessment group (BILAG) index. Serum levels of HMGB1 were analysed by western blot. HMGB1 expression in renal tissue was assessed by immunohistochemistry at baseline and follow-up biopsies in 25 patients.

Results

Baseline biopsies showed WHO class III, IV or V and all patients had high renal disease activity (BILAG A/B). Follow-up biopsies showed WHO I to II (n = 14), III (n = 6), IV (n = 3) or V (n = 12), and 15/35 patients were regarded as renal responders (BILAG C/D). At baseline HMGB1 was significantly elevated in serum compared to healthy controls (P < 0.0001). In all patients, serum levels decreased only slightly; however, in patients with baseline WHO class IV a significant decrease was observed (P = 0.03). Immunostaining revealed a pronounced extranuclear HMGB1 expression predominantly outlining the glomerular endothelium and in the mesangium. There was no clear difference in HMGB1 expression comparing baseline and follow-up biopsies or any apparent association to histopathological classification or clinical outcome.

Conclusions

Renal tissue expression and serum levels of HMGB1 were increased in LN. The lack of decrease of HMGB1 in serum and tissue after immunosuppressive therapy in the current study may reflect persistent inflammatory activity. This study clearly indicates a role for HMGB1 in LN.     

Recent Developments in the Role of High-Mobility Group Box 1 in Systemic Lupus Erythematosus

High-mobility group box 1 (HMGB1) is an important molecule for several nuclear processes. Recently, HMGB1 has gained much attention as a damage-associated molecular pattern (DAMP) and has been implicated in the pathogenesis of several (auto)-immune diseases, in particular, systemic lupus erythematosus (SLE). A main pathogenic feature in SLE is the accumulation of apoptotic cells. Since HMGB1 is released from apoptotic cells it has been hypothesized that HMGB1 might fuel the inflammatory processes, as seen in this disease, and play a fundamental role in the pathogenesis. In this review, we discuss evidence in support of the theory that HMGB1 is an important mediator in SLE and may be considered a new autoantigen.     

HMGB1 contributes to the development of acute lung injury after hemorrhage

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.     

High mobility group box 1 potentiates the pro-inflammatory effects of interleukin-1β in osteoarthritic synoviocytes

Introduction  

High mobility group box 1 (HMGB1) is released by necrotic cells or secreted in response to inflammatory stimuli. Extracellular HMGB1 may act as a pro-inflammatory cytokine in rheumatoid arthritis. We have recently reported that HMGB1 is released by osteoarthritic synoviocytes after activation with interleukin-1beta (IL-1β) The present study investigated the role of HMGB1 in synovial inflammation in osteoarthritis (OA).     

Systemic inflammation and remote organ injury following trauma require HMGB1

High-mobility group box 1 (HMGB1) is a 30-kDa DNA-binding protein that displays proinflammatory cytokine-like properties. HMGB1-dependent inflammatory processes have been demonstrated in models of sterile injury, including ischemia-reperfusion injury and hemorrhagic shock. Here, we tested the hypothesis that the systemic inflammatory response and associated remote organ injury that occur after peripheral tissue injury are highly dependent on HMGB1. Toll-like receptor 4 (TLR4) wild-type (WT) mice subjected to bilateral femur fracture after treatment with neutralizing antibodies to HMGB1 had lower serum IL-6 and IL-10 levels compared with mice treated with nonimmune control IgG. Similarly, compared with injured mice treated with control IgG, anti-HMGB1 antibody-treated mice had lower serum alanine aminotransferase levels and decreased hepatic and gut mucosal NF-kappaB DNA binding. TLR4 mutant (C3H/HeJ) mice subjected to bilateral femur fracture had less systemic inflammation and liver injury than WT controls. Residual trauma-induced systemic inflammation and hepatocellular injury were not ameliorated by treatment with a polyclonal anti-HMGB1 antibody, even though HMGB1 levels were transiently elevated just 1 h after injury in both WT and C3H/HeJ mice. Collectively, these data demonstrate a critical role for a TLR4-HMGB1 pathway in the initiation of systemic inflammation and end-organ injury following isolated peripheral tissue injury.     

HMGB1-dependent triggering of HIV-1 replication and persistence in dendritic cells as a consequence of NK-DC cross-talk

Background

HIV-1 has evolved ways to exploit DCs, thereby facilitating viral dissemination and allowing evasion of antiviral immunity. Recently, the fate of DCs has been found to be extremely dependent on the interaction with autologous NK cells, but the mechanisms by which NK-DC interaction controls viral infections remain unclear. Here, we investigate the impact of NK-DC cross-talk on maturation and functions of HIV-infected immature DCs.

Methodology/Principal Findings

Immature DCs were derived from primary monocytes, cultured in the presence of IL-4 and GM-CSF. In some experiments, DCs were infected with R5-HIV-1_BaL or X4-HIV-1_NDK, and viral replication, proviral HIV-DNA and the frequency of infected DCs were measured. Autologous NK cells were sorted and either kept unstimulated in the presence of suboptimal concentration of IL-2, or activated by a combination of PHA and IL-2. The impact of 24 h NK-DC cross-talk on the fate of HIV-1-infected DCs was analyzed. We report that activated NK cells were required for the induction of maturation of DCs, whether uninfected or HIV-1-infected, and this process involved HMGB1. However, the cross-talk between HIV-1-infected DCs and activated NK cells was functionally defective, as demonstrated by the strong impairment of DCs to induce Th1 polarization of naïve CD4 T cells. This was associated with the defective production of IL-12 and IL-18 by infected DCs. Moreover, the crosstalk between activated NK cells and HIV-infected DCs resulted in a dramatic increase in viral replication and proviral DNA expression in DCs. HMGB1, produced both by NK cells and DCs, was found to play a pivotal role in this process, and inhibition of HMGB1 activity by glycyrrhizin, known to bind specifically to HMGB1, or blocking anti-HMGB1 antibodies, abrogated NK-dependent HIV-1 replication in DCs.

Conclusion

These observations provide evidence for the crucial role of NK-DC cross-talk in promoting viral dissemination, and challenge the question of the in vivo involvement of HMGB1 in the triggering of HIV-1 replication and replenishment of viral reservoirs in AIDS.     

A high-sensitivity method for detection and measurement of HMGB1 protein concentration by high-affinity binding to DNA hemicatenanes

Background

Protein HMGB1, an abundant nuclear non-histone protein that interacts with DNA and has an architectural function in chromatin, was strikingly shown some years ago to also possess an extracellular function as an alarmin and a mediator of inflammation. This extracellular function has since been actively studied, both from a fundamental point of view and in relation to the involvement of HMGB1 in inflammatory diseases. A prerequisite for such studies is the ability to detect HMGB1 in blood or other biological fluids and to accurately measure its concentration.

Methodology/Principal Findings

In addition to classical techniques (western blot, ELISA) that make use of specific anti-HMGB1 antibodies, we present here a new, extremely sensitive technique that is based on the fact that hemicatenated DNA loops (hcDNA) bind HMGB1 with extremely high affinity, higher than the affinity of specific antibodies, similar in that respect to DNA aptamers. DNA-protein complexes formed between HMGB1 and radiolabeled hcDNA are analyzed by electrophoresis on nondenaturing polyacrylamide gels using the band-shift assay method. In addition, using a simple and fast protocol to purify HMGB1 on the basis of its solubility in perchloric acid allowed us to increase the sensitivity by suppressing any nonspecific background. The technique can reliably detect HMGB1 at a concentration of 1 pg per microliter in complex fluids such as serum, and at much lower concentrations in less complex samples. It compares favorably with ELISA in terms of sensitivity and background, and is less prone to interference from masking proteins in serum.

Conclusion

The new technique, which illustrates the potential of DNA nanoobjects and aptamers to form high-affinity complexes with selected proteins, should provide a valuable tool to further investigate the extracellular functions of HMGB1 and its involvement in inflammatory pathologies.     

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.     

Increased expression of Mer tyrosine kinase in circulating dendritic cells and monocytes of lupus patients: correlations with plasma interferon activity and steroid therapy

Introduction

The requirement for the immunoregulatory Mer tyrosine kinase (Mer) for optimal removal of apoptotic cells prompted us to look at its expression in systemic lupus erythematosus (SLE), in which apoptotic cell clearance is abnormal. We compared the levels of expression of Mer in normal human subjects and in patients with SLE.

Methods

We used flow cytometry of isolated peripheral blood mononuclear cells to compare the levels of Mer on leukocyte subsets. We used a Mer-specific enzyme-linked immunosorbent assay (ELISA) to quantify soluble Mer (sMer) in plasmas.

Results

Monocytes, CD1c⁺ myeloid dendritic cells (mDCs), and plasmacytoid dendritic cells (pDCs) from both normal individuals and from SLE patients expressed Mer. In both normal and SLE patients, the CD14⁺⁺CD16⁺ subpopulation of monocytes expressed the highest levels of Mer, with somewhat lower levels on the CD14^intCD16⁺ population. Mer levels on CD1c⁺ mDCs and pDCs, and sMer levels in blood were increased in SLE patients compared with controls. In patients, Mer levels on CD14^intCD16⁺, CD14⁺⁺CD16^- monocytes, and CD1c⁺ dendritic cells correlated positively with type I interferon (IFN-I) activity detected in blood. In SLE patients treated with corticosteroids, Mer expression on monocytes correlated with prednisone dose, CD1c⁺ myeloid dendritic cells in patients treated with prednisone had higher levels of Mer expression than those in patients not receiving prednisone.

Conclusions

We found no global defect in Mer expression in lupus blood. In contrast, we observed increased levels of Mer expression in DC populations, which could represent a response to increased IFN-I in SLE patients. Enhanced Mer expression induced by corticosteroids may contribute to its beneficial effects in SLE.     

Essential roles of high-mobility group box 1 in the development of murine colitis and colitis-associated cancer

High-mobility group box 1 (HMGB1) is a nuclear factor released extracellularly as a proinflammatory cytokine. We measured the HMGB1 concentration in the sera of mice with chemically induced colitis (DSS; dextran sulfate sodium salt) and found a marked increase. Inhibition of HMGB1 by neutralizing anti-HMGB1 antibody resulted in reduced inflammation in DSS-treated colons. In macrophages, HMGB1 induces several proinflammatory cytokines, such as IL-6, which are regulated by NF-kappaB activation. Two putative sources of HMGB1 were explored: in one, bacterial factors induce HMGB1 secretion from macrophages and in the other, necrotic epithelial cells directly release HMGB1. LPS induced a small amount of HMGB1 in macrophages, but macrophages incubated with supernatant prepared from necrotic cells and containing large amounts of HMGB1 activated NF-kappaB and induced IL-6. Using the colitis-associated cancer model, we demonstrated that neutralizing anti-HMGB1 antibody decreases tumor incidence and size. These observations suggest that HMGB1 is a potentially useful target for IBD treatment and the prevention of colitis-associated cancer.     

Monoclonal anti-HMGB1 (high mobility group box chromosomal protein 1) antibody protection in two experimental arthritis models

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.     

Dipotassium Glycyrrhizate Inhibits HMGB1-Dependent Inflammation and Ameliorates Colitis in Mice

Background

High mobility group box-1 (HMGB1) is a DNA-binding protein that is released from injured cells during inflammation. Advances in targeting HMGB1 represent a major challenge to improve the treatment of acute/chronic inflammation.

Aim

This study is aimed at verifying whether the inhibition of HMGB1 through dipotassium glycyrrhizate (DPG) is a good strategy to reduce intestinal inflammation.

Methods

Human colon adenocarcinoma cell line, HT29, human epithelial colorectal adenocarcinoma, Caco2, and murine macrophage cell line, RAW 264.7, were cultured to investigate the effect of DPG on the secretion of HMGB1. Acute colitis was induced in C57BL/6 mice through administration of 3% dextran sodium sulphate (DSS); a combined treatment with DSS and 3 or 8 mg/kg/day DPG was used to investigate the effects of DPG on intestinal inflammation. Animals were euthanized at seventh day and colonic samples underwent molecular and histological analyses.

Results

DPG significantly reduces in vitro the release of HMGB1 in the extracellular matrix as well as expression levels of pro-inflammatory cytokines, TNF-alpha, IL-1beta and IL-6, by inhibiting HMGB1. Moreover, DPG significantly decreases the severity of DSS-induced colitis in mice. Murine colonic samples show decreased mRNA levels of pro-inflammatory cytokines TNF-alpha, IL-1beta and IL-6, as well as HMGB1 receptors, RAGE and TLR4. Finally, HMGB1, abundantly present in the feces of mice with DSS-induced colitis, is strongly reduced by DPG.

Conclusions

HMGB1 is an early pro-inflammatory cytokine and an active protagonist of mucosal gut inflammation. DPG exerts inhibitory effects against HMGB1 activity, significantly reducing intestinal inflammation. Thus, we reason that DPG could represent an innovative tool for the management of human intestinal inflammation.     

Aberrant CD200/CD200R1 expression and function in systemic lupus erythematosus contributes to abnormal T-cell responsiveness and dendritic cell activity

Introduction

CD200 is a type I transmembrane glycoprotein that can regulate the activation threshold of inflammatory immune responses, polarize cytokine production, and maintain immune homeostasis. We therefore evaluated the functional status of CD200/CD200 receptor 1 (CD200R1) interactions in subjects with systemic lupus erythematosus (SLE).

Methods

Serum CD200 level was detected by ELISA. The expression of CD200/CD200R1 by CD4⁺ T cells and dendritic cells (DCs) was examined by flow cytometry, and then compared between SLE patients and healthy controls. Peripheral blood mononuclear cells were stained with carboxyfluorescein diacetate succinimidyl ester and annexin V/propidium iodide for evaluation of the effect of CD200 on cell proliferation and apoptosis. In addition, the effect of CD200 on DC function was determined by transwell migration assay as well as by measurement of binding and phagocytosis of apoptotic cells.

Results

In SLE patients, the number of CD200⁺ cells and the level of soluble CD200 were significantly higher than in healthy controls, whereas the expression of CD200R1 by CD4⁺ T cells and DCs was decreased. Furthermore, the increased CD200 expression by early apoptotic cells contributed to their diminished binding and phagocytosis by DCs in SLE. Importantly, the engagement of CD200 receptor on CD4⁺ T cells with CD200-Fc fusion protein in vitro reduced the differentiation of T-helper type 17 cells and reversed the defective induction of CD4⁺CD25^highFoxP3⁺ T cells by transforming growth factor beta in SLE patients. Conversely, blockade of CD200-CD200R1 interaction with anti-CD200R1 antibody promoted CD4⁺ T-cell proliferation.

Conclusion

CD200 and CD200R1 expression and function are abnormal in SLE and may contribute to the immunologic abnormalities in SLE.