Endothelial E- and P-selectin expression in iNOS- deficient mice exposed to polymicrobial sepsis.

In vitro, nitric oxide (NO) decreases leukocyte adhesion to endothelium by attenuating endothelial adhesion molecule expression. In vivo, lipopolysaccharide-induced leukocyte rolling and adhesion was greater in inducible NO synthase (iNOS)-/- mice than in wild-type mice. The objective of this study was to assess E- and P-selectin expression in the microvasculature of iNOS-/- and wild-type mice subjected to acute peritonitis by cecal ligation and perforation (CLP). E- and P-selectin expression were increased in various organs within the peritoneum of wild-type animals after CLP. This CLP-induced upregulation of E- and P-selectin was substantially reduced in iNOS-/- mice. Tissue myeloperoxidase (MPO) activity was increased to a greater extent in the gut of wild-type than in iNOS-/- mice subjected to CLP. In the lung, the reduced expression of E-selectin in iNOS-/- mice was not associated with a decrease in MPO. Our findings indicate that NO derived from iNOS plays an important role in sepsis-induced increase in selectin expression in the systemic and pulmonary circulation. However, in iNOS-/- mice, sepsis-induced leukocyte accumulation is affected in the gut but not in the lungs.

Role of endotoxin in the expression of endothelial selectins after cecal ligation and perforation.

The objectives of this study were to determine 1) the changes in endothelial cell adhesion molecule expression that occur in a clinically relevant model of sepsis and 2) the dependence of these changes on endotoxin [lipopolysaccharide (LPS)]. The dual radiolabeled monoclonal antibody technique was used to quantify the expression of E- and P-selectin in LPS-sensitive (C3HeB/FeJ) and LPS-insensitive (C3H/HeJ) mice that were subjected to acute peritonitis by cecal ligation and perforation (CLP). At 6 h after CLP, the expression of both E- and P-selectin was increased in the gut (mesentery, pancreas, and small and large bowel) compared with the sham-operated and/or control animals, with a more marked response noted in LPS-insensitive mice. The lung also exhibited an increased P-selectin expression in both mouse strains. An accumulation of granulocytes, assessed using tissue myeloperoxidase activity, was noted in the lung and intestine of LPS-sensitive but not LPS-insensitive mice exposed to CLP. These results indicate that the CLP model of sepsis is associated with an upregulation of endothelial selectins in the gut vasculature and that enteric LPS does not contribute to this endothelial cell activation response.

Microvascular dysfunction in sepsis.

The microvascular dysfunction which occurs in sepsis involves all three elements of the microcirculation: arterioles, capillaries, and venules. In sepsis, the arterioles are hyporesponsive to vasoconstrictors and vasodilators. Sepsis also reduces the number of perfused capillaries, thereby impacting on oxygen diffusion to mitochondria. In the venules of some tissues (e.g., mesentery) there is an inflammatory response characterized by neutrophil infiltration and protein leakage. In addition, PMN-endothelial adhesive interactions occur in precapillary microvessels and capillaries in organs, such as, the lung and heart. Thus, all these elements of the microcirculation are involved in the sepsis-induced inflammation. In this review we address emerging views on the mechanisms involved in the microvascular dysfunction induced by sepsis within the framework of these three basic elements of the microcirculatory unit.

LPS tolerance in human endothelial cells: reduced PMN adhesion, E-selectin expression, and NF-kappaB mobilization.

Cytokine release from inflammatory (CD14(+)) cells is reduced after repeated stimulation with lipopolysaccharide (LPS; LPS tolerance). However, it is not known whether LPS tolerance can be induced in CD14(-) cells. The aim of the present study was to determine whether endothelial cells [human umbilical vein endothelial cells (HUVEC)] could be rendered tolerant to LPS with respect to LPS-induced polymorphonuclear neutrophil (PMN) adhesion. LPS stimulation (0.5 microg/ml; 4 h) of naive HUVEC increased PMN adhesion. Pretreatment of HUVEC with LPS (0.5 microg/ml) for 24 h resulted in a reduction in the proadhesive effects of a subsequent LPS challenge. The initial LPS stimulation increased 1) mobilization of the nuclear transcription factor NF-kappaB to the nucleus and 2) surface levels of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and E-selectin. In LPS-tolerant HUVEC, a second LPS challenge resulted in 1) less accumulation of NF-kappaB in the nucleus, 2) a reduction in E-selectin expression, and 3) unchanged ICAM-1 expression. LPS-tolerant cells were still capable of mobilizing NF-kappaB in response to stimulation with either interleukin-1beta or tumor necrosis factor-alpha, resulting in elevated E-selectin levels and increased PMN adhesion. These studies show for the first time that LPS tolerance can be induced in endothelial cells with respect to PMN adhesion. This tolerance is specific for LPS and is associated with an inability of LPS to mobilize NF-kappaB, resulting in less E-selectin expression.

Anoxia/reoxygenation-induced tolerance with respect to polymorphonuclear leukocyte adhesion to cultured endothelial cells. A nuclear factor-kappaB-mediated phenomenon.

Exposing human umbilical vein endothelial cells (HUVECs) to anoxia/reoxygenation (A/R) results in an increase in polymorphonuclear leukocyte (PMN) adhesion to HUVECs. This A/R-induced hyperadhesion is completely prevented by a previous (24 hours earlier) exposure of HUVECs to A/R. This phenomenon has been termed "A/R tolerance." Exposing HUVECs to A/R induces an increase in nuclear factor kappaB (NF-kappaB) in HUVEC nuclei within 4 hours. Interfering with either NF-kappaB activation (proteasome inhibitor) or translocation (double-stranded oligonucleotides containing NF-kappaB binding sequence) prevents the development of A/R tolerance (ie, the increase in A/R-induced PMN adhesion to HUVECs is the same after the first and second A/R challenges). NO production by HUVECs is increased after the second A/R challenge, but not after the first A/R challenge. Inhibition of NO synthase (NOS) during the second A/R challenge prevents the development of A/R tolerance with respect to PMN adhesion. However, while HUVECs contained endothelial NOS protein, no inducible NOS was detected in either tolerant or nontolerant cells. Further studies indicated that inhibition of GTP-cyclohydrolase I (an enzyme involved in de novo synthesis of an important cofactor for NOS activity, tetrahydrobiopterin) prevented the generation of NO in A/R-tolerant cells. Extracellular generation of NO (NO donor) did not effect the hyperadhesion response induced by the initial A/R challenge. A/R also induced an oxidant stress in naive HUVECs, but not in A/R-tolerant HUVECs. Inhibition of NOS during the second A/R insult results in the generation of an oxidant stress similar to that observed after the first A/R challenge. Taken together, the findings of the present study are consistent with a role for NF-kappaB in the development of A/R tolerance (with respect to PMN adhesion), perhaps by transcriptional regulation of GTP-cyclohydrolase. The increased NO production during the second A/R insult reduces PMN adhesion most likely by reducing the intracellular oxidant stress induced by A/R.

Role of endonuclease activity and DNA fragmentation in Ca2+ ionophore A23187-mediated injury to rabbit isolated gastric mucosal cells.

In the current study, the role of endonuclease activity in calcium ionophore A23187-induced gastric mucosal cellular disruption was examined using rabbit gastric mucosal cells. Cell integrity was assessed using trypan blue dye exclusion and Alamar blue dye absorbance. Ionophore A23187 (1.6-25 microM) induced a concentration-dependent decrease in dye exclusion and cell metabolism in cells suspended in a medium containing Ca2+ (2 mM), while no such effect was observed in cells incubated in the absence of extracellular Ca2+. Cells that were pretreated with the endonuclease inhibitors aurintricarboxylic acid (ATCA; 0.2 or 0.5 mM or Zn2+; 0.01 and 0.1 mM) exhibited significant reduction in the total extent of cell injury when incubated with A23187 in the presence of Ca2+. DNA fragmentation as assessed by measurement of [3H]thymidine liberation or gel electrophoresis was increased in response to ionophore A23187 (12.5 or 25 microM) treatment. A minimal degree of fragmentation was observed when cells were suspended in a Ca(2+)-free medium or incubated in the presence of ATCA or Zn2+. Addition of ethanol (8% w/v) induced a significant increase in cell injury, which was not affected by either removal of extracellular Ca2+ or ATCA pretreatment. Furthermore, treatment with the antioxidants catalase (50 micrograms/ml) or 2',2'-dipyridyl (2 mM) reduced ionophore-induced cell injury but did not reduce the extent of DNA fragmentation. These data suggest that sustained increases in intracellular Ca2+ result in increased endonuclease activity in gastric mucosal cells, leading to extensive DNA lysis and cell damage. Ethanol-induced cell damage does not involve Ca2+ influx and therefore is not mediated by endonuclease activation. Furthermore, sustained increases in cellular Ca2+ may also mediate their effects via formation of reactive oxygen metabolites, but this mechanism of cell damage does not appear to involve DNA fragmentation.