Compartment syndrome causes systemic inflammation in a rat.

AIMS: Compartment syndrome results from increased intra-compartmental pressure (ICP) causing local tissue ischaemia and cell death, but the systemic effects are not well described. We hypothesised that compartment syndrome would have a profound effect not only on the affected limb, but also on remote organs. METHODS: Using a rat model of compartment syndrome, its systemic effects on the viability of hepatocytes and on inflammation and circulation were directly visualised using intravital video microscopy. RESULTS: We found that hepatocellular injury was significantly higher in the compartment syndrome group (192 PI-labelled cells/10(-1) mm(3), standard error of the mean (sem) 51) compared with controls (30 PI-labelled cells/10(-1) mm(3), sem 12, p < 0.01). The number of adherent venular white blood cells was significantly higher for the compartment syndrome group (5 leukocytes/30s/10 000 µm(2), sem 1) than controls (0.2 leukocytes/30 s/10 000 µm(2), sem 0.2, p < 0.01). Volumetric blood flow was not significantly different between the two groups, although there was an increase in the heterogeneity of perfusion. CONCLUSIONS: Compartment syndrome can be accompanied by severe systemic inflammation and end organ damage. This study provides evidence of the relationship between compartment syndrome in a limb and systemic inflammation and dysfunction in a remote organ. Cite this article: Bone Joint J 2016; 98-B:1132-7.

Contribution of inflammation to cellular injury in compartment syndrome in an experimental rodent model.

Compartment syndrome, a devastating consequence of limb trauma, is characterised by severe tissue injury and microvascular perfusion deficits. We hypothesised that leucopenia might provide significant protection against microvascular dysfunction and preserve tissue viability. Using our clinically relevant rat model of compartment syndrome, microvascular perfusion and tissue injury were directly visualised by intravital video microscopy in leucopenic animals. We found that while the tissue perfusion was similar in both groups (38.8% (standard error of the mean (sem) 7.1), 36.4% (sem 5.7), 32.0% (sem 1.7), and 30.5% (sem 5.35) continuously-perfused capillaries at 45, 90, 120 and 180 minutes compartment syndrome, respectively versus 39.2% (sem 8.6), 43.5% (sem 8.5), 36.6% (sem 1.4) and 50.8% (sem 4.8) at 45, 90, 120 and 180 minutes compartment syndrome, respectively in leucopenia), compartment syndrome-associated muscle injury was significantly decreased in leucopenic animals (7.0% (sem 2.0), 7.0%, (sem 1.0), 9.0% (sem 1.0) and 5.0% (sem 2.0) at 45, 90, 120 and 180 minutes of compartment syndrome, respectively in leucopenia group versus 18.0% (sem 4.0), 23.0% (sem 4.0), 32.0% (sem 7.0), and 20.0% (sem 5.0) at 45, 90, 120 and 180 minutes of compartment syndrome in control, p = 0.0005). This study demonstrates that the inflammatory process should be considered central to the understanding of the pathogenesis of cellular injury in compartment syndrome.

Traumatic injury elicits JNK-mediated human astrocyte retraction in vitro.

Brain injury causes dysfunction of the blood-brain barrier (BBB). The BBB is comprised of perivascular astrocytes whose end-feet ensheath brain microvascular endothelial cells. We investigated trauma-induced morphological changes of human astrocytes (HA) and human cerebral microvascular endothelial cells (hCMEC/D3) in vitro, including the potential role of mitogen-activated protein kinase (MAPK) signal-transduction pathways. HA or hCMEC/D3 were grown on flexible culture membranes and subjected to single traumatic injury normalized to 20%, 30% or 55% membrane deformation. Cells were assayed for morphological changes (i.e. retraction) and MAPK phosphorylation and/or expression (c-Jun NH2-terminal kinase (JNK)1/2, extracellular signal-regulated kinase (ERK)1/2, and p38). HA retraction was rapidly elicited with a single traumatic injury (55% membrane deformation; p<0.01). Morphological recovery of HA was observed within 2h (p<0.05). Traumatic injuries increased phospho-JNK1/2 (p<0.05) in HA, indicating MAPK activation. Pre-treatment of HA with structurally distinct JNK inhibitors (25µM), either SP600125 or SU3327, reduced JNK phosphorylation (p<0.05) and trauma-induced HA retraction (P<0.05). In contrast to HA, traumatic injury failed to induce either morphological changes or MAPK activation in hCMEC/D3. In summary, traumatic injury induces JNK-mediated HA retraction in vitro, while sparing morphological changes in cerebral microvascular endothelial cells. Astrocyte retraction from microvascular endothelial cells in vivo may occur after brain trauma, resulting in cellular uncoupling and BBB dysfunction. JNK may represent a potential therapeutic target for traumatic brain injuries.

Lung-derived mediators induce cytokine production in downstream organs via an NF-κB-dependent mechanism.

In the setting of acute lung injury, levels of circulating inflammatory mediators have been correlated with adverse outcomes. Previous studies have demonstrated that injured, mechanically ventilated lungs represent the origin of the host inflammatory response; however, mechanisms which perpetuate systemic inflammation remain uncharacterized. We hypothesized that lung-derived mediators generated by mechanical ventilation (MV) are amplified by peripheral organs in a "feed forward" mechanism of systemic inflammation. Herein, lung-derived mediators were collected from 129X1/SVJ mice after 2 hours of MV while connected to the isolated perfused mouse lung model setup. Exposure of liver endothelial cells to lung-derived mediators resulted in a significant increase in G-CSF, IL-6, CXCL-1, CXCL-2, and MCP-1 production compared to noncirculated control perfusate media (P < 0.05). Furthermore, inhibition of the NF-κB pathway significantly mitigated this response. Changes in gene transcription were confirmed using qPCR for IL-6, CXCL-1, and CXCL-2. Additionally, liver tissue obtained from mice subjected to 2 hours of in vivo MV demonstrated significant increases in hepatic gene transcription of IL-6, CXCL-1, and CXCL-2 compared to nonventilated controls. Collectively, this data demonstrates that lung-derived mediators, generated in the setting of MV, are amplified by downstream organs in a feed forward mechanism of systemic inflammation.

Mediators released from LPS-challenged lungs induce inflammatory responses in liver vascular endothelial cells and neutrophilic leukocytes.

The systemic inflammatory response plays an important role in the progression of acute lung injury (ALI) to multiple organ dysfunction syndrome (MODS). However, the role of lung-derived inflammatory mediators in induction of the inflammatory response in remote organs is poorly understood. To address the above, we investigated the effects of lung inflammation on induction of inflammatory response(s) in the liver in vitro. Inflammation in mouse lungs was induced by intranasal administration of lipopolysaccharide (LPS; 1 mg/ml) followed by mechanical ventilation using the isolated perfused mouse lung method to obtain and characterize lung perfusate from the pulmonary circulation. LPS administration to mouse lungs resulted in an increased release of inflammation-relevant cytokines and chemokines into the perfusate (Luminex assay) compared with the saline-controls. Subsequently, primary mouse liver vascular endothelial cells (LVEC) or mouse polymorphonuclear leukocytes (PMN) in vitro were stimulated with the perfusate obtained from saline- or LPS-challenged lungs and assessed for various inflammation-relevant end points. The obtained results indicate that stimulation of LVEC with perfusate obtained from LPS-challenged lungs results in 1) reactive oxygen species (ROS) production; 2) activation of NF-kappaB; and 3) expression of E-selectin, ICAM-1, and VCAM-1 and a subsequent increase in PMN rolling and adhesion to LVEC. In addition, perfusate from LPS-challenged lung induced activation of PMN with respect to increased ROS production and upregulation of cell surface levels of adhesion molecules MAC-1 and VLA-4. Heat-inactivation of the perfusate obtained from LPS-challenged lungs was very effective in suppressing increased proadhesive phenotype (i.e., E-selectin and ICAM-1 expression) in LVEC, whereas targeted inhibition (immunoneutralization) of TNF-alpha and/or IL-6 in LPS-lung perfusate had no effect. Taken together, these findings indicate that multiple proinflammatory mediators (proteinaceous in nature) released from inflamed lungs act synergistically to induce systemic activation of circulating PMN and promote inflammatory responses in liver vascular endothelial cells.

Dextran sulfate sodium-induced acute colonic inflammation in angiotensin II type 1a receptor deficient mice.

OBJECTIVE: Angiotensin II (Ang II) receptor blockers have been reported to contribute to cytoprotective effects in various organs. However, the role of renin-angiotensin system (RAS) in modulation of the inflammatory bowel disease (IBD) remains unclear. In this study we assessed the role of angiotensin II type 1a (AT1a) receptor on the outcome of dextran sulfate sodium (DSS)-induced acute colitis by employing AT1a receptor deficient mice. MATERIALS AND METHODS: The acute colitis was induced in wild type (WT) and AT1a receptor deficient mice by giving orally 3% DSS in drinking water for 7 days. RESULTS: Induction of DSS colitis resulted in up-regulation of Ang II and AT1a receptor in the colonic mucosa of WT mice. In parallel, loss of body weight, an increase in disease activity index (DAI), and the shortening of colon were found in DSS-challenged WT mice. In addition, an increase in thiobarbituric acid (TBA)-reactive substances and myeloperoxidase (MPO) activity, along with the up-regulation of tumor necrosis factor (TNF)-alpha were detected in the colonic mucosa of DSS-challenged WT mice. The endpoints mentioned above were significantly ameliorated in DSS-challenged AT1a receptor deficient mice. CONCLUSIONS: RAS is involved in the pathophysiology of DSS-induced colitis and AT1a receptor may be a novel therapeutic target for the treatment of IBD.

Neutrophil-endothelial cell interactions during the development of tolerance to ischaemia/reperfusion in isolated cells.

Ischaemia/reperfusion (I/R) tolerance refers to the phenomenon by which the inflammation and associated sequelae induced by I/R is ameliorated by an I/R challenge imposed 24 h earlier. The development of I/R tolerance is dependent on the synthesis of new proteins. In vivo and in vitro studies provide support for nitric oxide synthase (NOS), antioxidant enzymes, and heat shock proteins (HSPs) as the effector proteins. Activation of the nuclear transcription factor, NFkappaB, appears to be a prerequisite for the development of I/R tolerance. In vitro approaches using anoxia/reoxygenation (A/R) to mimic I/R have provided insights into the complexity of the development of I/R tolerance, i.e. different cells may use different signalling pathways to develop A/R tolerance and influence the responses of adjacent cells during the process. The use of cells from genetically altered mice is expediting attempts to unravel specific mechanisms involved in the development of A/R tolerance.

Peritonitis induces rat cardiac myocytes to promote polymorphonuclear leukocyte emigration and activate endothelial cells: effect of lipopolysaccharide pretreatment.

OBJECTIVE: Peritonitis induced by cecal ligation and perforation results in inflammation and dysfunction of the rat myocardium, an organ remote from the locus of infection. This peritonitis-induced pathology can be prevented by pretreating these animals with lipopolysaccharide before cecal ligation and perforation. In the present study, we assessed a) whether cardiomyocytes obtained from rats subjected to cecal ligation and perforation could induce polymorphonuclear leukocyte transendothelial migration, b) whether these cardiomyocytes could activate endothelial cells (increased proadhesive phenotype), and c) whether these responses could be attenuated by lipopolysaccharide pretreatment. DESIGN: Prospective animal study. SETTING: Experimental animal laboratory. SUBJECTS: Male Sprague Dawley rats. INTERVENTIONS: Lipopolysaccharide pretreated and nonpretreated rats were subjected to cecal ligation and perforation or to laparotomy. Myocytes were isolated 6 hrs after surgery and used for in vitro experiments. MEASUREMENTS AND MAIN RESULTS: Myocytes isolated from cecal ligation and perforation rats promoted migration of polymorphonuclear leukocytes across a rat endothelial cell monolayer, an effect prevented by platelet activating factor receptor antagonists. Myocytes isolated from these animals also increased surface level expression of intercellular adhesion molecule-1 on rat endothelial cells, an effect also prevented by platelet activating factor receptor antagonists. Myocytes isolated from rats pretreated with lipopolysaccharide and then subjected to cecal ligation and perforation did not a) promote polymorphonuclear leukocyte transendothelial migration or b) increase intercellular adhesion molecule-1 surface expression on endothelial cells. CONCLUSIONS: Our findings indicate that induction of peritonitis results in a systemic response that induces cardiac myocytes to become proinflammatory (i.e., these myocytes produce chemotactic factors and activate endothelial cells). This effect of cecal ligation and perforation is abrogated by pretreating animals with lipopolysaccharide before induction of peritonitis.

Cardiac myocytes exposed to anoxia-reoxygenation promote neutrophil transendothelial migration.

The goal of the present study was to assess whether cardiac myocytes exposed to anoxia-reoxygenation (A/R) could generate a chemotactic gradient for polymorphonuclear neutrophil (PMN) transendothelial migration. Exposure of neonatal mouse cardiac myocytes to A/R induced an oxidant stress in the myocytes. Supernatants obtained from A/R-conditioned myocytes promoted mouse PMN migration across mouse myocardial endothelial cell monolayers. This increase in PMN transendothelial migration could be prevented if catalase or a platelet-activating factor (PAF) antagonist was added to the supernatants before assay. Supernatants from A/R-conditioned myocytes activated endothelial cells by inducing an intracellular oxidant stress. The oxidant stress and PMN transendothelial migration induced by supernatants from A/R-conditioned myocytes were substantially reduced when endothelial cells derived from manganese superoxide dismutase overexpressing mice were used in the assays. Supernatants from A/R-conditioned myocytes also increased endothelial cell surface levels of E-selectin and intercellular adhesion molecule-1. Our results indicate that cardiac myocytes exposed to A/R can generate a chemotactic gradient, presumably due to production and release of stable oxidants and PAF. The ability of supernatants from A/R-conditioned myocytes to promote PMN transendothelial migration was largely dependent on induction of an oxidant stress in endothelial cells. In addition, these supernatants also induced a proadhesive phenotype in the endothelial cells.

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.

Nitric oxide attenuates but superoxide enhances iNOS expression in endotoxin- and IFNgamma-stimulated skeletal muscle endothelial cells.

OBJECTIVE: Endothelial cells (ECs) in septic skeletal muscle may be exposed to large amounts of NO and superoxide generated by the skeletal muscle cells. We tested the hypothesis that inducible nitric oxide synthase (iNOS) induction in ECs (i.e., one of the steps in the septic process) is modulated by extravascularly generated nitric oxide (NO) and superoxide. METHODS: To model sepsis in vitro, monolayers of microvascular ECs derived from rat skeletal muscle were incubated with a mixture of lipopolysaccharide (LPS) (25 ng/mL) and interferongamma (IFNgamma) (100 U/mL) for up to 24 hours. Next, a long-term release NO donor (DETA NONOate), a superoxide-generating mixture (xanthine oxidase/xanthine; XO/X), or DETA + XO/X were added to the LPS + IFNgamma mixture. The iNOS protein and activity, as well as intracellular oxidative stress, were measured at intervals up to 24 hours, whereas the activation of AP-1, IRF-1, and NFkappaB transcription factors was determined at 2 and 24 hours. RESULTS: LPS + IFNgamma caused time-dependent increases in iNOS protein and activity. Increasing concentrations of DETA (up to 500 microM) decreased, whereas XO/X (10 mU per mL/0.1 mM, respectively) markedly enhanced, iNOS expression and activity. DETA attenuated the enhancement by XO/X. Although intracellular oxidative stress was not altered by LPS + IFNgamma, modulations of iNOS expression by DETA, XO/X, and DETA + XO/X correlated with changes in oxidative stress. Among the three transcription factors, only IRF-1 and NFkappaB seemed to be involved in iNOS induction and its modulation by DETA and XO/X. CONCLUSIONS: LPS + IFNgamma can induce iNOS expression in microvascular ECs from rat skeletal muscle, whereas NO and superoxide modulate this expression. On the basis of these observations we suggest that NO and superoxide from the extravascular tissue may play a key role in the inflammatory response of septic ECs.

Endotoxin promotes adhesion of human erythrocytes to human vascular endothelial cells under conditions of flow.

OBJECTIVE: To investigate the effects of endotoxin on adhesion of human red blood cells to human vascular endothelial cells under conditions of flow. DESIGN: Prospective, randomized, controlled in vitro study. SETTINGS: University-affiliated cell biology laboratory. SUBJECTS: Human erythrocytes and human vascular endothelial cells. INTERVENTIONS: Fresh human erythrocytes and human vascular endothelial cells grown as monolayers were incubated with either saline or endotoxin. After incubation, endothelial monolayers were superfused with erythrocytes, and the number of erythrocytes adhering to the endothelial monolayer was quantified. MEASUREMENTS AND MAIN RESULTS: Adhesion of erythrocytes to vascular endothelium was measured under conditions of continuous flow in different settings: a) exposure of both endothelial cells and erythrocytes to saline; b) incubation of both erythrocytes and endothelial cells with endotoxin; c) exposure of erythrocytes only to endotoxin; d) incubation of endothelial cells only to endotoxin; and e) both the endothelial cells and erythrocytes incubated with different concentrations of endotoxin. Erythrocyte adhesion in the saline control group was 71 +/- 8 cells/mm2. Incubation of both components with endotoxin increased the number of adhesive erythrocytes to 172 +/- 9 cells/mm2 (p < .05). When only the endothelial cells were treated with endotoxin, 142 +/- 8 cells/mm2 adhered to the endothelial monolayer, whereas the incubation of the erythrocytes only to endotoxin resulted in adhesion of 102 +/- 3 cells/mm2. Decreasing concentrations of endotoxin reduced adhesion from 172 +/- 9 cells/mm2 (endotoxin, 75 microg/mL) to 165 +/- 9 cells/mm2 (endotoxin, 25 microg/mL), 153 +/- 4 cells/mm2 (endotoxin, 1 microg/mL), and 146 +/- 6.1 cells/mm2 (endotoxin, 5 ng/mL). CONCLUSIONS: Exposure of human erythrocytes and human venous vascular endothelial cells to an inflammatory stimulus such as endotoxin promotes a dose-dependent adhesion of erythrocytes to endothelium in a dynamic environment. These adhesive erythrocyte-endothelium interactions can be produced by exposure of either red blood cells or endothelial cells to endotoxin, with a higher degree of adhesion after activation of the endothelial cell component.

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.

LPS pretreatment ameliorates peritonitis-induced myocardial inflammation and dysfunction: role of myocytes.

Peritonitis induced by cecal ligation and puncture (CLP) produces a systemic inflammatory response that can be largely mitigated by pretreatment of the animals with lipopolysaccharide (LPS tolerance). Although cells of myeloid origin and endothelial cells have been shown to contribute to the development of LPS tolerance, little is known regarding the potential role of parenchymal cells in this phenomenon. The major aim of the present study was to assess whether cardiac parenchymal cells (myocytes) contribute to the development of LPS tolerance. Six hours after induction of CLP rats were neutropenic and acidotic, the myocardium contained a leukocyte infiltrate [myeloperoxidase (MPO) activity was increased], and myocardial contractile function was impaired (left ventricular developed pressure was decreased). In animals that were pretreated with LPS these manifestations of sepsis were largely reversed. Further studies focused on the responses of cardiac myocytes to CLP and whether myocytes contributed to the development of LPS tolerance. Myocytes were isolated from rat hearts 6 h after induction of CLP. These myocytes 1) exhibited an impaired ability to shorten in response to pacing, 2) contained the nuclear transcription factor NF-kappaB in their nuclei, 3) increased their surface levels of intercellular adhesion molecule-1 (ICAM-1), and 4) were hyperadhesive for neutrophils. All of these events did not occur in myocytes obtained from animals that were pretreated with LPS before induction of CLP. These findings indicate that LPS tolerance can be induced in myocytes with respect to polymorphonuclear leukocyte adhesion, presumably by an inability of CLP to mobilize NF-kappaB to the myocyte nuclei and, thereby, preventing an increase in surface levels of ICAM-1.

PAF-induced elastase-dependent neutrophil transendothelial migration is associated with the mobilization of elastase to the neutrophil surface and localization to the migrating front.

One of the cardinal signs of acute inflammation is neutrophil (PMN) emigration across the endothelium and into the affected tissue. We have previously shown that human PMN migration across human umbilical vein endothelial cell (HUVEC) monolayers is dependent on PMN-derived elastase. However, whether migrating PMN release elastase into the extracellular milieu or retain it on the cell surface is unclear. In the present study, we show that when PMN are activated by platelet activating factor (PAF), elastase was mobilized to and retained in the cell membrane; no elastase activity was detected in the supernatant. Neutroplasts (enucleated cells devoid of granules) prepared from PAF-activated PMN contained twice as much elastase as did neutroplasts prepared from unstimulated PMN. Neutroplasts from PAF-activated PMN migrated across HUVEC monolayers in response to a chemotactic gradient (PAF), while those prepared from unstimulated PMN did not. The neutroplast transendothelial migration was inhibited (80%) by a monoclonal antibody against elastase. Using confocal microscopy, we noted that the localization of elastase on the cell surface of PMN, which were adherent to HUVEC but not migrating, was largely confined to the apical aspect of the PMN. There was little or no elastase detectable on the basal aspect of the PMN membrane in contact with the endothelium. By contrast, in migrating PMN the membrane-bound elastase was primarily localized to the migrating front, i.e. pseudopodia penetrating the HUVEC monolayers. Taken together, our findings indicate that migrating PMN localize their membrane-bound elastase to the migrating front where it facilitates transendothelial migration.

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.

Transendothelial neutrophil migration. Role of neutrophil-derived proteases and relationship to transendothelial protein movement.

During an acute inflammatory response polymorphonuclear leukocytes (PMNs) adhere to and emigrate across the venular microvasculature. There is general agreement on the mechanisms involved in PMN adhesive interactions. However, the mechanisms by which PMNs migrate across the endothelial lining remain controversial, particularly with respect to the role of elastase. In the present study, we used human umbilical vein endothelial cells (HUVECs) and PMNs to test the hypothesis that the relative role of PMN-derived elastase may be dependent on the degree of HUVEC retraction within monolayers. A high (10(-7) mol/L), but not a low (10(-10) mol/L), concentration of platelet-activating factor (PAF) caused HUVEC retraction of sufficient magnitude to increase transendothelial protein movement. Elastase inhibitors prevented PMN transendothelial migration in response to the low, but not the high, concentration of PAF. These findings suggest that PMN migration across confluent endothelial cells is elastase dependent, whereas PMN migration across retracted endothelial cells is elastase independent. However, under the latter condition (high concentration of PAF), the two endogenous proteases, alpha 2-macroglobulin and alpha 1-antitrypsin, could interfere with PAF-induced PMN transendothelial migration. Thus, as the concentration of PAF is increased, migrating PMNs use other proteases, in addition to elastase. We also noted that transendothelial protein movement is closely coupled to PMN migration.

Extract of Helicobacter pylori induces neutrophils to injure endothelial cells and contains antielastase activity.

BACKGROUND & AIMS: Previous studies indicate that a water extract of Helicobacter pylori promotes leukocyte adhesion and emigration as well as endothelial barrier disruption (increased vascular protein leakage) in rat mesenteric venules. The aims of this study were to assess whether H. pylori extract-activated neutrophils disrupt endothelial cell monolayers and to identify the mechanisms involved in this process. METHODS: Human neutrophils were incubated with monolayers of human umbilical vein endothelial cells (HUVECs) in the presence or absence of H. pylori extract. RESULTS: H. pylori extract-activated human neutrophils produced endothelial cell detachment from HUVEC monolayers, the severity of which was dependent on the duration of exposure. Endothelial cell detachment was prevented by a monoclonal antibody directed against CD11/CD18 on neutrophils or a monoclonal antibody against intercellular adhesion molecule 1 on endothelial cells. HUVEC monolayer disruption was also prevented by superoxide dismutase, catalase, and a monoclonal antibody against elastase. Further studies indicated that H. pylori extract was capable of inhibiting human neutrophil elastase. The antielastase activity was not diminished by oxidants. CONCLUSIONS: These studies indicate that H. pylori extract-activated human neutrophils can disrupt HUVEC monolayers only when human neutrophils are allowed to adhere to HUVECs and may provide an explanation for the H. pylori extract-induced, neutrophil-dependent vascular protein leakage observed in vivo. The possibility that H. pylori releases antiproteases may explain, in part, why this bacterium is so virulent.

Regional differences in constitutive and induced ICAM-1 expression in vivo.

The aim of the present study was to characterize and compare the expression of intercellular adhesion molecule 1 (ICAM-1) on unstimulated and endotoxin-challenged endothelial cells in different tissues of the rat. ICAM-1 expression was measured using 125I-labeled anti-rat ICAM-1 monoclonal antibody (MAb) and an isotype-matched control MAb labeled with 131I (to correct for nonspecific accumulation of the binding MAb). Under baseline conditions, ICAM-1 MAb binding was observed in all organs. The binding of 125I-ICAM-1 MAb varied widely among organs, with the largest accumulation (per g tissue) in the lung, followed by heart (1/30th of lung activity), splanchnic organs (1/50th of lung activity), thymus (1/100th of lung activity), testes (1/300th of lung activity), and skeletal muscle (1/800th of lung activity). Endotoxin induced an increase in ICAM-1 MAb binding in all organs except the spleen. Endotoxin-induced upregulation of ICAM-1 was greatest in heart and skeletal muscle (5- to 10-fold), whereas the remaining organs exhibited a two- to fourfold increase in ICAM-1 expression. Maximal upregulation of ICAM-1 occurred at 9-12 h after endotoxin administration. A dose-dependent increase in ICAM-1 expression was elicited by 0.1-10 microgram/kg, with higher doses (up to 5 mg/kg) producing no further increment. Induction of ICAM-1 mRNA after endotoxin was observed in all tissues examined (lung, heart, intestine), peaked at 3 h, and then rapidly returned to control levels. These findings indicate that ICAM-1 is constitutively expressed on vascular endothelium in all organs of the rat and that there are significant regional differences in the magnitude and time course of endotoxin-induced ICAM-1 expression.

Aspirin-induced, neutrophil-mediated injury to vascular endothelium.

Previous studies indicate that aspirin can promote neutrophil (PMN) adhesion to endothelial cells and neutrophil-mediated endothelial cell detachment. The objectives of the present study were to determine whether PMN adhesion is a prerequisite for aspirin-induced, PMN-mediated endothelial cell detachment and whether neutrophil-derived oxidants and/or proteases are responsible for the cell detachment. Human PMNs were added to confluent monolayers of human umbilical vein endothelial cells (HUVEC) and coincubated with or without aspirin at a clinically relevant concentration (300 micrograms/ml). Aspirin-activated PMNs induced endothelial cell detachment, but not cell lysis. Endothelial cell detachment was always preceded by retraction of endothelial cells within the monolayer. The aspirin-induced, neutrophil-mediated cell detachment was prevented by a monoclonal antibody directed against CD11/CD18 adhesion integrins on PMNs. Elastase inhibitors, but not superoxide dismutase or catalase, prevented both endothelial cell retraction and detachment. If aspirin-activated neutrophils were allowed to migrate across the monolayers, endothelial cell retraction or detachment did not occur. These studies indicate that aspirin-induced, PMN-mediated endothelial cell retraction and detachment requires PMN adhesion to the target cells and is due to neutrophil-derived elastase. Endothelial cell retraction, induced by activated neutrophils, may represent an exaggeration of a normal physiologic event, i.e., neutrophil emigration.