Activation of human umbilical vein endothelial cells leads to relocation and release of high-mobility group box chromosomal protein 1.

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)-alpha. 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-alpha 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.

High mobility group 1 B-box mediates activation of human endothelium.

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-kappaB. 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.

Evidence for an abnormal profile of interleukin-4 (IL-4), IL-5, and gamma-interferon (gamma-IFN) in peripheral blood T cells from patients with allergic eosinophilic gastroenteritis.

Allergic eosinophilic gastroenteritis is characterized by elevated total IgE, specific IgE to food antigens, and eosinophilia of tissue and blood. Because the lymphokines IL-4, IL-5, and gamma-interferon, regulate IgE synthesis, and eosinophilopoiesis in vitro, we examined whether there is an imbalance in their production in allergic eosinophilic gastroenteritis. To explore this hypothesis, three adult patients with allergic eosinophilic gastroenteritis were studied. Flow cytometric studies of peripheral blood mononuclear cells from these patients did not reveal evidence of T cell activation or disturbance of T cell numbers or subsets. T cells were capable of normal mitogenic activation in vitro. IL-4 and IL-5 production were markedly elevated with mitogenic stimulation. Most IL-4 and IL-5 production was by CD4+ T cells. Synthesis of IL-5 by CD4+ T lymphocytes in three patients and CD8+ T lymphocytes in two patients occurred in the absence of mitogen. Mitogen-stimulated GM-CSF and gamma-interferon synthesis by CD4+ T cells was normal. Lymphokine mRNA in total cellular RNA derived from endoscopic biopsies was examined by reverse transcription/polymerase chain reaction. Mucosal biopsies from control subjects and most biopsies from allergic eosinophilic gastroenteritis patients contained less than 10(-8) micrograms IL-5 mRNA/1 microgram total cellular mRNA. gamma-Interferon mRNA was not detected by reverse transcription/polymerase chain reaction in biopsies from patients with allergic eosinophilic gastroenteritis but was present in controls. These lymphokine abnormalities are consistent with the elevated IgE and eosinophilia seen in allergic eosinophilic gastroenteritis and suggest that strategies targeting T lymphocytes may be efficacious in treatment of this disease.