Regional sympathetic blockade attenuates activation of intestinal macrophages and reduces gut barrier failure.

BACKGROUND: Endotoxin-induced activation of monocytes may lead to extravasation of cells, excessive production of nitric oxide, and subsequent epithelial injury in the gut. Regional sympathetic blockade by means of thoracic epidural anesthesia has been implicated to protect the epithelial barrier. This study tested the hypothesis that thoracic epidural anesthesia decreases epithelial permeability by attenuating monocytic production of nitric oxide and nitrosative stress. METHODS: Rats were anesthetized, hemodynamically monitored, and mechanically ventilated. Endotoxemia was induced by an intravenous bolus injection of Escherichia coli lipopolysaccharide. Either lidocaine 2% or normal saline was injected as a bolus, followed by a continuous infusion via an epidural catheter. Three hundred minutes after injection of lipopolysaccharide or normal saline, gut epithelial permeability to fluorescein isothiocyanate-dextran (4 kDa), intestinal expression of inducible nitric oxide synthase by macrophages, and lipid peroxidation represented by 8-isoprostane tissue concentration were quantified. RESULTS: Thoracic epidural anesthesia significantly attenuated the endotoxin-induced increases in gut epithelial permeability (437 [293, 492] vs. 628 [532, 1,042] ng/ml, median [quartiles], P = 0.03), expression of nitric oxide synthase (2 [1,2] vs. 7 [5,8] cells per 384 µm(2), P = 0.003), macrophage infiltration, and lipid peroxidation (22,460 ± 11,476 vs. 37,840 ± 17,551 pg/ml, mean ± SD, P = 0.05). CONCLUSIONS: Thoracic epidural anesthesia attenuates endotoxin-induced gut epithelial injury. This is likely due to a decrease in monocytic extravasation and intestinal nitrosative stress. As possible mechanisms, direct nerve-immune interplay, a reduction in plasma catecholamines, or a systemic lidocaine effect has to be considered.

Adrenomedullin reduces intestinal epithelial permeability in vivo and in vitro.

Leakage of the gut mucosal barrier in the critically ill patient may allow translocation of bacteria and their virulence factors, thereby perpetuating sepsis and inflammation. Present evidence suggests that adrenomedullin (AM) improves endothelial barrier function and stabilizes circulatory function in systemic inflammation. We tested the hypothesis that exogenously applied AM stabilizes gut epithelial barrier function. Infusion of Staphylococcus aureus alpha-toxin induced septic shock in rats. AM infusion in a therapeutic setting reduced translocation of labeled dextran from the gut into the systemic circulation in this model. AM also reduced alpha-toxin and hydrogen peroxide (H2O2)-related barrier disruption in Caco-2 cells in vitro and reduced H2O2-related rat colon barrier malfunction in Ussing chamber experiments. AM was shown to protect endothelial barrier function via cAMP elevation, but AM failed to induce cAMP accumulation in Caco-2 cells. cAMP is degraded via phosphodiesterases (PDE), and Caco-2 cells showed high activity of cAMP-degrading PDE3 and 4. However, AM failed to induce cAMP accumulation in Caco-2 cells even in the presence of sufficient PDE3/4 inhibition, whereas adenylyl cyclase activator forskolin induced strong cAMP elevation. Furthermore, PDE3/4 inhibition neither amplified AM-induced epithelial barrier stabilization nor affected AM cAMP-related rat colon short-circuit current, furthermore indicating that AM may act independently of cAMP in Caco-2 cells. Finally, experiments using chemical inhibitors indicated that PKC, phosphatidylinositide 3-kinase, p38, and ERK did not contribute to AM-related stabilization of barrier function in Caco-2 cells. In summary, during severe inflammation, elevated AM levels may substantially contribute to the stabilization of gut barrier function.

Adrenomedullin reduces vascular hyperpermeability and improves survival in rat septic shock.

OBJECTIVE: Current therapies of sepsis and septic shock require administration of a large volume of fluid to maintain hemodynamic stability. The vasoregulatory peptide adrenomedullin has been shown to prevent the transition to the fatal hypocirculatory septic state by poorly understood mechanisms. We tested the hypothesis that therapeutic administration of adrenomedullin would reduce vascular hyperpermeability, thereby contributing to improved hemodynamics and survival. DESIGN: Prospective randomized controlled animal study. SUBJECTS: Male Sprague-Dawley rats (270 g). INTERVENTIONS: We used 4.8 x 10(3) U/kg of Staphylococcus aureus alpha-toxin, a pore-forming exotoxin, to induce vascular leakage and circulatory shock in rats. The infusion rate was 24 microg/kg per hour. Adrenomedullin was started 1 h after alpha-toxin administration. MEASUREMENT AND RESULTS: Infusion of alpha-toxin in rats induced cardiocirculatory failure resulting in a 6-h mortality of 53%. alpha-Toxin provoked massive vascular hyperpermeability, which was indicated by an enrichment of Evans blue dye albumin in the tissues of lung, liver, ileum and kidney. Plasma fluid loss led to a significant hemoconcentration. Hemodynamic impairment observed after alpha-toxin infusion was closely correlated to vascular hyperpermeability. Therapeutic administration of 24 microg/kg per hour adrenomedullin reduced 6-h mortality from 53% to 7%. Stabilization of the endothelial barrier by adrenomedullin was indicated by reduced extravasation of albumin and plasma fluid and may have contributed to hemodynamic improvement. CONCLUSIONS: These data suggest that adrenomedullin-related reduction of vascular hyperpermeability might represent a novel and important mechanism contributing to the beneficial effects of this endogenous vasoregulatory peptide in sepsis and septic shock.

Perturbation of endothelial junction proteins by Staphylococcus aureus alpha-toxin: inhibition of endothelial gap formation by adrenomedullin.

Endothelial hyperpermeability is a hallmark of an inflammatory reaction and contributes to tissue damage in severe infections. Loss of endothelial cell-cell adhesion leads to intercellular gap formation allowing paracellular fluid flux. Although Staphylococcus aureus alpha-toxin significantly contributed to staphylococci disease, little is known about its mechanism of endothelial hyperpermeability induction. Here, we demonstrate that in a model of isolated perfused rat ileum discontinuation of capillary vascular-endothelial-cadherin (VE-cadherin) was observed after bolus application of S. aureus alpha-toxin being inhibited by the endogenous peptide adrenomedullin (ADM). In vitro, alpha-toxin exposure induced loss of immunoreactivity of VE-cadherin and occludin in human cultured umbilical vein endothelial cells. Likewise, ADM blocked alpha-toxin-related junctional protein disappearance from intercellular sites. Additionally, cyclic AMP elevation was shown to stabilize endothelial barrier function after alpha-toxin application. Although no RhoA activation was noted after endothelial alpha-toxin exposure, inhibition of Rho kinase and myosin light chain kinase blocked loss of immunoreactivity of VE-cadherin and occludin as well as intercellular gap formation. In summary, stabilization of endothelial junctional integrity as indicated by interendothelial immunostaining might be an interesting approach to stabilize endothelial barrier function in severe S. aureus infections.

Regulation of interleukin IL-4, IL-13, IL-10, and their downstream components in lipopolysaccharide-exposed rat lungs. Comparison of the constitutive expression between rats and humans.

Interleukin (IL)-4, IL-10, and IL-13 are T-helper2 cell derived cytokines, which are known to suppress pro-inflammatory cytokine production. The biologically related IL-4 and IL-13 have an impact on the development of atopic inflammation, whereas IL-10 is mostly supposed to be involved in fibrotic disorders or inflammatory bowel disease. Their influence on the pathogenesis of severe lung injury is widely unknown. The expression of these proteins is mostly assumed to be restricted to leukocytic cells. Recently, some non-leukocytic cell types have been described to generate these mediators. In the present study, the constitutive cellular distribution pattern of IL-4, IL-13, IL-10, IL-4R alpha, STAT6 and IL-10R was elaborated by immunohistochemistry in the rat organism. Cytokine-specific regulation in lipopolysaccharide (LPS)-challenged rat lungs was investigated and constitutive expression was compared with human lungs. The study demonstrates strong expression of IL-4, IL-10, and IL-13 in different non-leukocytic cell types, especially in endothelial and epithelial cells in the entire rat organism. In concert with rat lung expression human lungs show strong similarities. Moreover, vascular LPS challenge of rat lungs generally demonstrates cell type-specific downregulation of the cytokines. We conclude that non-leukocytic cells in the organism play an important role in the regulation of organ-specific inflammatory reactions, especially in lungs.

Adrenomedullin treatment abolishes ileal mucosal hypoperfusion induced by Staphylococcus aureus alpha-toxin--an intravital microscopic study on an isolated rat ileum.

OBJECTIVE: Disturbances of intestinal microcirculation associated with sepsis and septic shock result in diminished mucosal oxygenation. Tissue hypoxia as well as mediator formation may lead to intestinal mucosa dysfunction. As a consequence, bacteria and their products as well as gut-derived inflammatory mediators may further perpetuate septic and inflammatory events. Adrenomedullin is produced in the mucosa of the gastrointestinal tract and has been shown to improve survival in experimental sepsis. Using pore-forming Staphylococcus aureus alpha-toxin as a potent initiator of inflammatory reactions, we tested the hypothesis that exogenously added adrenomedullin improves ileal mucosal perfusion. DESIGN: Prospective, experimental study. SETTING: University laboratory. SUBJECTS: Isolated perfused ileum from male Sprague-Dawley rats INTERVENTIONS: Adrenomedullin treatment of S. aureus alpha-toxin infused ileum. MEASUREMENT AND MAIN RESULTS: An infusion of alpha-toxin (0.05 microg/mL) induced a significant decrease of red blood cell velocity in villus terminal arterioles from 1.7 to 0.7 mm/sec assessed by intravital microscopy. This was accompanied by a significant reduction of mucosal hemoglobin oxygenation from 71.8% to 17.5% and impaired oxygen uptake. At constant bulk flow and oxygen delivery, these data indicate a redistribution of blood perfusion away from mucosa. Subsequent intervention with 0.1 microM adrenomedullin redistributed blood flow back toward the mucosa, causing an improvement of mucosal hemoglobin oxygenation and of organ oxygen uptake. CONCLUSION: These data suggest that exogenously added adrenomedullin protects ileum mucosa by diminishing alpha-toxin-induced microcirculatory disturbances. Further investigations will have to clarify the therapeutic potential of adrenomedullin in sepsis-related gut dysfunction.

Streptococcus pneumoniae R6x induced p38 MAPK and JNK-mediated caspase-dependent apoptosis in human endothelial cells.

Streptococcus pneumoniae is the major pathogen of community-acquired pneumonia and a common cause of otitis, meningitis and sepsis. During pneumococci infection accompanied with bacterial invasion and hematogenous spreading, the endothelium is directly targeted by pneumococci and their virulence factors. Therefore, we tested the hypothesis that pneumococci induced endothelial apoptosis. Unencapsulated R6x pneumococci strongly induced apoptosis of human endothelial cells both from lung microvasculature and umbilical vein, whereas an encapsulated strain D39 mainly led to necrotic cell death. Deletion of the gene coding for pneumolysin reduced pneumococci-induced apoptosis in HUVEC. Furthermore, N-acetyl-L-cysteine, an antioxidant thiol, significantly reduced apoptosis caused by R6x, and LDH release induced by D39, pointing to a role for reactive oxygen species in the pathogenesis. Apoptotic cells showed increased cleavage and activity of caspases 6 and 9 but only late activation of caspase 3. Programmed cell death could be strongly reduced by pan-caspase inhibitor zVAD. Reduced levels of Bcl2 and cytosolic increase of apoptosis-inducing factor in pneumococci-infected cells implicated involvement of mitochondrial death pathways. Caspase activation and apoptosis were abolished by cAMP elevation. Moreover, p38 mitogen-activated protein kinase and c-Jun NH(2)-terminal kinase were activated in pneumococci-infected cells and inhibitors of both kinases strongly reduced pneumococci-induced caspase activation and apoptosis. Hence, kinase- and caspase-dependence of pneumococci-induced endothelial apoptosis may bear relevance to novel therapeutic approaches to pneumococci-related disease.

Adrenomedullin reduces Staphylococcus aureus alpha-toxin-induced rat ileum microcirculatory damage.

OBJECTIVE: Increased microvascular permeability and perfusion mismatch are hallmarks of sepsis or septic shock. The intestinal mucosa is very sensitive to tissue hypoxia. Intestinal mucosa dysfunction may allow translocation of bacteria and their products, thereby perpetuating sepsis and inflammation. Staphylococcus aureus alpha-toxin is a major pathogenicity determinant of this bacterium, provoking cardiovascular collapse. Current evidence suggests that the endogenous peptide adrenomedullin stabilizes circulatory homeostasis in systemic inflammatory response. Using alpha-toxin as a well-defined strong initiator of an inflammatory reaction, we tested the hypothesis that exogenously applied adrenomedullin stabilizes gut microcirculation. DESIGN: Prospective, experimental study. SETTING: Research laboratory at a university hospital. SUBJECTS: Isolated, perfused ileum from male Sprague-Dawley rats and human umbilical vein endothelial cells. INTERVENTIONS: Administration of S. aureus alpha-toxin before or after infusion of adrenomedullin. MEASUREMENTS AND MAIN RESULTS: Injection of a bolus of 1 microg of alpha-toxin in the superior mesenteric artery in a constant-flow, blood-perfused preparation of rat ileum increased perfusion pressure and relative hemoglobin concentration and decreased mucosal hemoglobin oxygen saturation. Continuous infusion of adrenomedullin (0.1 micromol/L) significantly reduced these alpha-toxin-related effects. Severe microvascular hyperpermeability observed in alpha-toxin-exposed ileum was abolished by adrenomedullin pretreatment. In addition, adrenomedullin blocked alpha-toxin-induced endothelial myosin light chain phosphorylation, endothelial cell contraction, and subsequent loss of endothelial barrier function in vitro. Treatment of alpha-toxin (infusion of 0.05 microg/mL)-exposed ileum with adrenomedullin (0.1 micromol/L) started 10 mins after onset of toxin application also significantly reduced superior mesenteric artery pressure and permeability increase. CONCLUSIONS: In summary, these data suggest that exogenous adrenomedullin protects ileum by reducing alpha-toxin-induced microcirculatory disturbances and by stabilizing endothelial barrier function.