To study the systemic release and kinetics of high mobility group box-1 protein (HMGB1) in relation to clinical features in a population of patients with severe sepsis or septic shock and to compare these with the kinetics of the cytokines interleukin-6, interleukin-8, interleukin-10, and tumor necrosis factor-α. Prospective study of two cohorts of patients. Intensive care unit and infectious disease clinic at Karolinska University Hospital Huddinge. Twenty-six patients with severe sepsis, 33 patients with septic shock, and a reference group of five patients with sepsis. None. Sixty-four patients were included, ten of whom died within 28 days. Cytokine levels were measured at five time points during the first week after admission and were correlated to Acute Physiology and Chronic Health Evaluation II and Sepsis-related Organ Failure Assessment scores. Two HMGB1 assays were used. Both demonstrated delayed kinetics for HMGB1 with high levels on inclusion that remained high throughout the study period. Serum concentration at 144 hrs, the last sampling point, was 300 times higher, 34,000 ± 76,000 pg/mL (mean ± sd), than any of the other cytokines. This study, however, found no predictable correlation between serum levels of HMGB1 and severity of infection. We did quite unexpectedly find significantly lower levels of HMGB1 in nonsurvivors compared with survivors as measured by our main assay, but the other showed no difference between the two groups. Levels of interleukin-6, interleukin-8, interleukin-10, and tumor necrosis factor-α correlated significantly with severity of disease, and all were significantly higher in patients with septic shock compared with those with severe sepsis. Neither of these comparisons showed significant correlations for HMGB1. This is the first prospective study assessing the release over time of HMGB1 in a population of patients with sepsis, severe sepsis, or septic shock. Levels remained high in the majority of patients up to 1 wk after admittance, indicating that the cytokine indeed is a downstream and late mediator of inflammation. Further studies are required to fully define the relationship of HMGB1 to severity of disease.

Treutiger CJ, Mullins GE, Johansson A‐SM, Rouhiainen A, Rauvala HME, Erlandsson‐Harris H, Andersson U, Yang H, Tracey KJ, Andersson J, Palmblad JEW (Center for Infectious Medicine; Center for Inflammation and Haematology Research; Karolinska Institute at Huddinge University Hospital, Stockholm, Sweden, Finnish Red Cross Blood Transfusion Service; Institute of Biotechnology, University of Helsinki; Helsinki, Finland, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden and North Shore‐Long Island Jewish Research Institute, NY, USA). High mobility group 1 B‐box mediates activation of human endothelium. J Intern Med 2003; 254: 375–385.Objectives. Severe sepsis and septic shock is a consequence of a generalized inflammatory systemic response because of an invasive infection that may result in acute organ dysfunction. Mortality is high despite access to modern intensive care units. The nuclear DNA binding protein high mobility group 1 (HMGB1) protein has recently been suggested to act as a late mediator of septic shock via its function as a macrophage‐derived pro‐inflammatory cytokine (J Exp Med 2000; 192: 565, Science1999; 285: 248). We investigated the pro‐inflammatory activities of the A‐box and the B‐box of HMGB1 on human umbilical venular endothelial cells (HUVEC).Design. The HUVEC obtained from healthy donors were used for experiments. Recombinant human full‐length HMGB1, A‐box and B‐box were cloned by polymerase chain reaction (PCR) amplification from a human brain quick‐clone cDNA. The activation of HUVEC was studied regarding (i) upregulation of adhesion molecules, (ii) the release of cytokines and chemokines, (iii) the adhesion of neutrophils to HUVEC, (iv) the activation of signalling transduction pathways and (v) the involvement of the receptor for advanced glycation end‐products (RAGE).Results. The full‐length protein and the B‐box of HMGB1 dose‐dependently activate HUVEC to upregulate adhesion molecules such as ICAM‐1, VCAM‐1 and E‐selectin and to release IL‐8 and G‐CSF. The activation of HUVEC could be inhibited to 50% by antibodies directed towards the RAGE. HMGB1‐mediated HUVEC stimulation resulted in phosphorylation of the ELK‐1 signal transduction protein and a nuclear translocation of p65 plus c‐Rel, suggesting that HMGB1 signalling is regulated in endothelial cells through NF‐κB.Conclusions. The HMGB1 acts as a potent pro‐inflammatory cytokine on HUVEC and the activity is mainly mediated through the B‐box of the protein. HMGB1 may be a key factor mediating part of the pro‐inflammatory response occurring in septic shock and severe inflammation.

Amphoterin (HMGB1) is a 30-kD heparin-binding protein involved in process extension and migration of cells by a mechanism involving the receptor for advanced glycation end products (RAGE). High levels of amphoterin are released to serum during septic shock. We have studied the expression of amphoterin in monocytes and the role of amphoterin and RAGE in monocyte transendothelial migration. Un-activated monocytes in suspension did not reveal amphoterin on their surface, but adherent monocytes exported amphoterin to the cell surface. Immunohistochemical staining of arterial thrombi in vivo revealed amphoterin in mononuclear cells and in surrounding extracellular matrix. Amphoterin was secreted from phorbol ester and interferon-γ (IFN-γ)-activated macrophages, and the secretion was inhibited by blocking the adenosine 5′-triphosphate (ATP)-binding cassette transporter-1, a member of the multidrug resistance protein family. Amphoterin was specifically adhesive for monocytes in peripheral blood leukocyte adhesion assay. Adhesion caused an extensive spreading of cells, which was inhibited by the dominant-negative RAGE receptor (soluble ectodomain of RAGE), and adhesion up-regulated chromogranin expression in monocytes, also suggesting a RAGE-dependent interaction. Monocyte transendothelial migration was efficiently inhibited by anti-amphoterin and anti-RAGE antibodies and by the soluble RAGE. We suggest that amphoterin is an autocrine/paracrine regulator of monocyte invasion through the endothelium. (Blood. 2004;104:1174-1182)

HMGB1 (amphoterin) is a 30-kDa heparin-binding protein that mediates transendothelial migration of monocytes and has proinflammatory cytokine-like activities. In this study, we have investigated proinflammatory activities of both highly purified eukaryotic HMGB1 and bacterially produced recombinant HMGB1 protens. Mass analyses revealed that recombinant eukaryotic HMGB1 has an intrachain disulphide bond. In mass analysis of tissue-derived HMGB1, two forms were detected: the carboxyl terminal glutamic acid residue lacking form and a full-length form. Cell culture studies indicated that both eukaryotic and bacterial HMGB1 proteins induce TNF-α secretion and nitric oxide release from mononuclear cells. Affinity chromatography analysis revealed that HMGB1 binds tightly to proinflammatory bacterial substances. A soluble proinflammatory substance was separated from the bacterial recombinant HMGB1 by chloroform-methanol treatment. HMGB1 interacted with phosphatidylserine in both solid-phase binding and cell culture assays, suggesting that HMGB1 may regulate phosphatidylserine-dependent immune reactions. In conclusion, HMGB1 polypeptide has a weak proinflammatory activity by itself, and it binds to bacterial substances, including lipids, that may strengthen its effects.

Ordered differential display identified a novel sequence induced in neurons by the neurite-promoting protein amphoterin. We named this gene amphoterin-induced gene and ORF (AMIGO), and also cloned two other novel genes homologous to AMIGO (AMIGO2 and AMIGO3). Together, these three AMIGOs form a novel family of genes coding for type I transmembrane proteins which contain a signal sequence for secretion and a transmembrane domain. The deduced extracellular parts of the AMIGOs contain six leucine-rich repeats (LRRs) flanked by cysteine-rich LRR NH₂- and COOH-terminal domains and by one immunoglobulin domain close to the transmembrane region. A substrate-bound form of the recombinant AMIGO ectodomain promoted prominent neurite extension in hippocampal neurons, and in solution, the same AMIGO ectodomain inhibited fasciculation of neurites. A homophilic and heterophilic binding mechanism is shown between the members of the AMIGO family. Our results suggest that the members of the AMIGO protein family are novel cell adhesion molecules among which AMIGO is specifically expressed on fiber tracts of neuronal tissues and participates in their formation.

The nuclear protein high‐mobility group box chromosomal protein 1 (HMGB1) was recently described to act as a pro‐inflammatory cytokine and as a late mediator of severe sepsis and septic shock. The protein is released from monocytes in response to endotoxin and activates monocytes and endothelial cells through nuclear factor kappa B. We have previously demonstrated that the B‐box of HMGB1 mediates a pro‐inflammatory effect on endothelial cells including the upregulation of cell‐adhesion molecules and release of interleukin (IL)‐8 and granulocyte colony‐stimulating factor. Here, we report that HMGB1 is released from human umbilical vein endothelial cells (HUVEC) in response to lipopolysaccharide (LPS) and tumour necrosis factor (TNF)‐α. A nuclear relocation of HMGB1 to the cytoplasm was seen at 4 h. Subsequently, high amounts of HMGB1 could be seen in the supernatants from stimulated cells after 16 h. It was also observed that the pro‐inflammatory activity of HMGB1 is sensitive to dexamethasone. Interestingly, the HMGB1‐induced TNF‐α release from monocytes could be inhibited by either the A‐box of the protein or the p38 inhibitor CNI‐1493, but neither had any inhibitory effects on the HMGB1‐dependent upregulation of cell‐adhesion molecules on HUVEC. Altogether, these results suggest that HUVEC may be an important source of HMGB1 secretion in response to systemic infection and that endothelial cells and monocytes may use different signalling pathways.