Virginia S. Carl

Gut ischemia/reperfusion produces lung injury independent of endotoxin

ObjectiveBacterial translocation from the gut has been invoked as a common inciting event for postinjury multiple organ failure. We previously showed that gut ischemia/reperfusion induces remote organ injury. The purpose of this study was to ascertain if endotoxin has a pivotal mechanistic role in this process. DesignProspective, randomized study. SettingAnimal laboratory. SubjectsSprague-Dawley rats weighing 300 to 350 g. InterventionsAnesthetized animals underwent 45 mins of superior mesenteric artery occlusion and 2 hrs of reperfusion; sham laparotomy served as controls. Endotoxin was eliminated with the murine immunoglobulin (Ig) M antibody E5, 3 mg/kg iv before the study. Measurements and Main ResultsPlasma endotoxin was measured by the limulus amebocyte lysate assay. At 2 hrs of reperfusion, circulating neutrophil priming was determined by the difference in superoxide generation with and without the activating stimulus, N-formyl-Met-Leu-Phe. Neutrophil sequestration in the lung was quantitated by myeloperoxidase activity, and by lung endothelial permeability by 125I albumin lung/blood ratio. Endotoxin concentrations were not significantly (significance determined as p < .05) different between the gut ischemia/reperfusion and laparotomy groups (n = ≥5) during ischemia or reperfusion. Circulating neutrophil priming, neutrophil accumulation in the lung, and lung injury were provoked by gut ischemia/reperfusion, but not altered by endotoxin elimination. ConclusionGut ischemia/reperfusion primes circulating neutrophils and produces lung injury by a mechanism independent of endotoxin. (Crit Care Med 1994; 22:1438–1444)

Interleukin-8 increases endothelial permeability independent of neutrophils.

Interleukin-8 (IL-8) has been associated with a variety of hyperinflammatory states and adverse clinical events. Circulating IL-8 levels correlate with the severity of tissue trauma, and excessive elevations of IL-8 are associated with postinjury adult respiratory distress syndrome and multiple organ failure. While IL-8 is a potent neutrophil (PMN) chemoattractant and activator and enhances PMN transendothelial migration, it also acts to inhibit PMN adhesion to stimulated endothelial cells (ECs). We hypothesized that IL-8 could interact directly with ECs to increase permeability independent of PMNs. Human umbilical vein ECs (HUVECs) were cultured on collagen-coated micropore filters, and integrity of the EC monolayer measured by albumin flux across the filter. Cytochalasin D was used as a positive control. IL-8 induced increased permeability at a concentration of 1000 ng/mL. This effect was abrogated by preincubation of HUVECs with a protein synthesis inhibitor (cycloheximide). These data suggest a role for IL-8 in promoting endothelial leak independent of PMNs, via a mechanism involving protein synthesis.

Involvement of bradykinin B1 and B2 receptors in human PMN elastase release and increase in endothelial cell monolayer permeability

Bradykinin (BK) is a potent inflammatory mediator, which can release other inflammatory mediators by interacting with bradykinin B1 and B2 receptors. The role of kinins in regulating human PMN elastase release was studied. BK induced elastase release 5-fold over basal levels. Elastase release was inhibited by both B1 and B2 receptor antagonists. A specific B1 agonist des-Arg10-KD increased elastase release 4-fold. Since elastase has been implicated in vascular leak, the effect of BK on endothelial cell monolayer (EM) permeability was assessed. BK increased EM leak (I125 flux) across the EM, whereas des-Arg10-KD was inactive. When co-cultured with human umbilical vein endothelial cells, des-Arg10-KD-treated PMNs increased EM leak by 35%. The elastase inhibitor AAVPK blocked des-Arg10-KD-induced leak by 80% suggesting that elastase is responsible for the increase in permeability. It is concluded that BK causes increased leak by inducing PMN elastase release via activation of both B1 and B2 receptors. BK blockade and elastase inhibition may be beneficial in inflammatory diseases such as ARDS which is characterized by increased lung permeability and both kinin and PMN activation are thought to participate.

Intercellular adhesion molecule—1 promotes neutrophil-mediated cytotoxicity*

BACKGROUND Interaction of the CD11/CD18 complex on polymorphonuclear neutrophils (PMNs) and intercellular adhesion molecule (ICAM)-1 on endothelium is a critical event in PMN-mediated tissue injury. In addition, increased expression of ICAM-1 on type I pneumocytes has been identified in a variety of pulmonary disorders associated with PMN-induced inflammation. We hypothesized that ICAM-1 up-regulation is sufficient to promote cytotoxicity via activated PMNs. METHODS The complementary DNA for human ICAM-1 was transfected into Chinese hamster ovarian (CHO) cells, which do not inherently express this adhesion receptor, by using the expression vector CD1.8. Fluorescence-activated cell sorter analysis revealed 62% CHO cell surface expression of ICAM-1. Wild type and transfected CHO cells were labeled with chromium 51 and exposed to quiescent or activated (1 mumol/L phorbol myristate acetate) PMNs for 4 hours. Subsets were pretreated with a monoclonal antibody to ICAM-1. PMN cytotoxicity was determined by specific percent 51Cr release. RESULTS Incubation of quiescent PMNs with wild type and transfected CHO cells produced nominal cell lysis, 0.5% +/- 0.3% and 0.2% +/- 0.2%, respectively. Activated PMNs produced 13.6% +/- 3.2% versus 1.4% +/- 0.7% cell lysis, comparing transfected with wild type CHO cells, and 0.5% +/- 0.2% cell lysis after pretreatment with a monoclonal antibody to ICAM-1, p < 0.01. CONCLUSIONS ICAM-1 up-regulation is sufficient to promote cytotoxicity via activated PMNs. This may represent a potential target for attenuating PMN-mediated injury to endothelial and other cell lines, including parenchyma.

Lipopolysaccharide-induced CD11B-mediated neutrophil-endothelial adhesion is not required for polymorphonuclear cell priming.

Previous work has implicated both neutrophil-endothelial cell (PMN-EC) adhesion and PMN priming (enhanced superoxide production following activation) in the development of postinjury adult respiratory distress syndrome (ARDS) and multiple organ failure (MOF). CD11B, a member of the integrin family of PMN surface receptors, has been alleged to have a prominent role in these inflammatory PMN-EC processes. The purpose of the present study was to test the hypothesis that CD11B-mediated PMN-EC adhesion is necessary for endotoxin (LPS)-induced PMN priming. Human neutrophils, isolated by Percoll gradient centrifugation, were exposed to LPS (100 ng/mL). At fixed times over 120 minutes (a) superoxide following fMLP activation (i.e., priming), (b) PMN-EC adhesion, and (c) expression of CD11B were assayed. Superoxide production was measured by cytochrome c reduction, PMN-EC adhesion with indium-labelled PMN adherence to human umbilical vein endothelial cell (HUVEC) monolayer cultures, and CD11B expression with fluorescent labelled anti-CD11B (60.1) antibodies. The PMN-EC adhesion was biphasic, with an early maximum at 15 minutes followed by a nadir at 60 minutes and secondary rise through 120 minutes of LPS exposure. CD11B expression changed dramatically in temporal association with early PMN-EC adhesion, but the secondary increase in adhesion was associated with only a mild rise in CD11B expression. PMN priming increased after a latency of 15 minutes to a maximum of 800 nmol/10(6) cells/min after 60 minutes of LPS exposure.(ABSTRACT TRUNCATED AT 250 WORDS)