Transcriptional arrest of the human E-selectin gene.

BACKGROUND: E-selectin transcription requires binding of transcription factors, NF-kappaB, ATF-2, and HMG-I(Y). Here we characterize the mechanism responsible for the transcriptional downregulation of E-selectin expression. MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with TNF-alpha for 24 h. HUVEC E-selectin expression was measured by enzyme-linked immunosorbent assay, Northern blotting, and nuclear run-on assays, and NF-kappa B was assessed by electrophoretic gel mobility shift assays (EMSAs). RESULTS: (1) E-selectin surface expression peaked at 4 h and then diminished over the next 20 h. (2) Transcription of E-selectin began within 1 h of TNF-alpha exposure and ceased by 8 h, despite continuous stimulation of HUVECs with TNF-alpha. (3) EMSAs revealed persistent binding activity of NF-kappa B proteins to two NF-kappa B-binding sites during 24 h of continuous stimulation with TNF-alpha. However, binding activity of proteins that recognize a third NF-kappa B element, -126 to -116 bp from the transcription start site, was lost after 4 h during 24 h of continuous stimulation with TNF-alpha; ATF-2 binding was unchanged over 24 h stimulation with TNF-alpha. CONCLUSION: The termination of E-selectin expression is controlled at the level of transcription, with loss of protein-DNA interactions at only one of three NF-kappa B-binding sites in the E-selectin promoter.

Hypothermia inhibits human E-selectin transcription.

During endothelial cell activation, the formation and expression of E-selectin require transcriptional activation of the E-selectin gene, mediated by the coordinated action of several transcription factors and cis-acting elements in its 5'-flanking region. It is reported that in vitro hypothermia (25 degrees C) transiently inhibits transcriptional activation and surface expression of E-selectin as well as neutrophil adherence to cultured human umbilical vein endothelial cells (HUVECs) treated with lipopolysaccharide (LPS), interleukin-1 (IL-1), or tumor necrosis factor (TNF). Rewarming HUVECs treated with LPS, IL-1, or TNF to 37 degrees C restores E-selectin transcript accumulation, E-selectin surface expression, and neutrophil adherence to HUVECs at levels equivalent to similarly treated HUVECs maintained at 37 degrees C continuously. Despite the absence of detectable E-selectin transcription at 25 degrees C, activation of the transcription factor NF-kappaB still occurred in HUVECs treated with LPS, IL-1, or TNF, indicating that signal transduction was not blocked by hypothermia. It is concluded that neutrophil adherence to activated endothelium mediated by E-selectin is reversibly inhibited by hypothermia. The protective effect of hypothermia clinically (e.g., cardiopulmonary bypass) may, in part, be mediated by transiently inhibiting the expression of an endothelial cell activation phenotype.

Activation of human endothelial cells by viable or heat-killed gram-negative bacteria requires soluble CD14.

In response to bacterial lipopolysaccharides (LPS; endotoxin), endothelial cells are converted to an activation phenotype expressing both proinflammatory and procoagulant properties that include the induction of leukocyte adhesion molecules and tissue factor expression. LPS-induced endothelial cell activation requires a soluble form of the monocyte LPS receptor, sCD14. We evaluated the capacity of multiple strains of gram-negative and gram-positive bacteria to induce endothelial E-selectin and tissue factor expression through sCD14-dependent pathways with cultured human umbilical vein endothelial cells (HUVE). Both viable and heat-killed gram-negative bacteria (Bacteroides fragilis, Enterobacter cloacae, Haemophilus influenzae, and Klebsiella pneumoniae) but not viable or heat-killed gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis, and Streptococcus pneumoniae) induced prominent E-selectin surface expression detected by enzyme-linked immunosorbent assay. Tissue factor activity on HUVE, indicated by factor X activation, was induced in response to gram-negative bacteria but not in response to gram-positive bacteria. Gram-negative bacteria induced transcriptional activation in HUVE, indicated by the appearance of E-selectin-specific mRNA and by the demonstration of activation of NF-kappa B, a trans-activating factor necessary for E-selectin and tissue factor gene transcription. In contrast, neither E-selectin mRNA nor activation of NF-kappa B was detected in HUVE treated with gram-positive bacteria. Endothelial cell activation by gram-negative bacteria in each of these assays was inhibited with a monoclonal antibody (60bd) against CD14. Furthermore, CHO-K1 cells, transfected with human recombinant CD14, responded to all strains of gram-negative bacteria (viable or heat killed), indicated by CHO-K1 NF-kappa B activation. We conclude that gram-negative bacteria induce endothelial cell activation through a common sCD14-dependent pathway.

Small bowel resection for relief of chronic intestinal pseudo-obstruction.

Chronic intestinal pseudo-obstruction refractory to medical therapy is a debilitating problem for patients and a challenge for clinicians. We report a case of chronic idiopathic intestinal pseudo-obstruction with giant upper intestinal diverticula, complicated by hypersecretion refractory to medical therapy and requiring 10 yr of home parenteral nutrition. Resection of the chronically dilated small bowel with giant diverticula and construction of improved gastric and duodenal drainage was performed. This not only relieved the hypersecretory state and improved gastric drainage, but allowed the patient to stop parenteral nutrition and eat regular food for the first time in 10 yr. Select patients with chronic intestinal pseudo-obstruction will respond favorably to palliative surgical intervention.