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Objectives: Severe pneumonia may evoke acute lung injury, and sphingosine-1-phosphate is involved in the regulation of vascular permeability and immune responses. However, the role of sphingosine-1-phosphate and the sphingosine-1-phosphate producing sphingosine kinase 1 in pneumonia remains elusive. We examined the role of the sphingosine-1-phosphate system in regulating pulmonary vascular barrier function in bacterial pneumonia., Design: Controlled, in vitro, ex vivo, and in vivo laboratory study., Subjects: Female wild-type and SphK1-deficient mice, 8-10 weeks old. Human postmortem lung tissue, human blood-derived macrophages, and pulmonary microvascular endothelial cells., Interventions: Wild-type and SphK1-deficient mice were infected with Streptococcus pneumoniae. Pulmonary sphingosine-1-phosphate levels, messenger RNA expression, and permeability as well as lung morphology were analyzed. Human blood-derived macrophages and human pulmonary microvascular endothelial cells were infected with S. pneumoniae. Transcellular electrical resistance of human pulmonary microvascular endothelial cell monolayers was examined. Further, permeability of murine isolated perfused lungs was determined following exposition to sphingosine-1-phosphate and pneumolysin., Measurements and Main Results: Following S. pneumoniae infection, murine pulmonary sphingosine-1-phosphate levels and sphingosine kinase 1 and sphingosine-1-phosphate receptor 2 expression were increased. Pneumonia-induced lung hyperpermeability was reduced in SphK1 mice compared with wild-type mice. Expression of sphingosine kinase 1 in macrophages recruited to inflamed lung areas in pneumonia was observed in murine and human lungs. S. pneumoniae induced the sphingosine kinase 1/sphingosine-1-phosphate system in blood-derived macrophages and enhanced sphingosine-1-phosphate receptor 2 expression in human pulmonary microvascular endothelial cell in vitro. In isolated mouse lungs, pneumolysin-induced hyperpermeability was dose dependently and synergistically increased by sphingosine-1-phosphate. This sphingosine-1-phosphate-induced increase was reduced by inhibition of sphingosine-1-phosphate receptor 2 or its downstream effector Rho-kinase., Conclusions: Our data suggest that targeting the sphingosine kinase 1-/sphingosine-1-phosphate-/sphingosine-1-phosphate receptor 2-signaling pathway in the lung may provide a novel therapeutic perspective in pneumococcal pneumonia for prevention of acute lung injury.

Objectives: Community-acquired pneumonia is a very common infectious disease associated with significant morbidity and mortality. Streptococcus pneumoniae is the predominant pathogen in this disease, and pneumococcal resistance to multiple antibiotics is increasing. The recently purified bacteriophage endolysin Cpl-1 rapidly and specifically kills pneumococci on contact. The aim of this study was to determine the therapeutic potential of Cpl-1 in a mouse model of severe pneumococcal pneumonia., Design: Controlled, in vivo laboratory study., Subjects: Female C57/Bl6 mice, 8-12 weeks old., Interventions: Mice were transnasally infected with pneumococci and therapeutically treated with Cpl-1 or amoxicillin by intraperitoneal injections starting 24 or 48 hours after infection., Measurements and Main Results: Judged from clinical appearance, decreased body weight, reduced dynamic lung compliance and Pao2/Fio2 ratio, and morphologic changes in the lungs, mice suffered from severe pneumonia at the onset of therapy. When treatment was commenced 24 hours after infection, 100% Cpl-1-treated and 86% amoxicillin-treated mice survived otherwise fatal pneumonia and showed rapid recovery. When treatment was started 48 hours after infection, mice had developed bacteremia, and three of seven (42%) Cpl-1-treated and five of seven (71%) amoxicillin-treated animals survived. Cpl-1 dramatically reduced pulmonary bacterial counts, and prevented bacteremia, systemic hypotension, and lactate increase when treatment commenced at 24 hours. In vivo, treatment with Cpl-1 or amoxicillin effectively reduced counts of penicillin-susceptible pneumococci. The inflammatory response in Cpl-1-and amoxicillin-treated mice was lower than in untreated mice, as determined by multiplex cytokine assay of lung and blood samples. In human epithelial cell cultures, lysed bacteria evoked less proinflammatory cytokine release and cell death, as compared with viable bacteria., Conclusions: Cpl-1 may provide a new therapeutic option in the treatment of pneumococcal pneumonia.

Objective: Acute respiratory failure is a significant complication of severe pneumococcal pneumonia. In a mouse model, we observed early-onset lung microvascular leakage after pulmonary infection with Streptococcus pneumoniae, and we hypothesized that the important virulence factor pneumolysin may be the direct causative agent., Design: Controlled, in vivo, ex vivo, and in vitro laboratory study., Setting: Laboratory., Subjects: Female mice, 8-12 wks old., Interventions: Ventilated and blood-free perfused murine lungs were challenged with recombinant pneumolysin via the airways as well as via the vascular bed. In addition, we analyzed the impact of pneumolysin on electrical cell impedance and hydraulic conductivity of human umbilical vein endothelial cell (HUVEC) and alveolar epithelial cell (A549) monolayers., Measurements and Main Results: Aerosolized pneumolysin dose-dependently increased capillary permeability with formation of severe lung edema but did not affect pulmonary vascular resistance. Intravascular pneumolysin caused an impressive dose-dependent increase in pulmonary vascular resistance and in lung microvascular permeability. By immunohistochemistry, pneumolysin was detected mainly in endothelial cells of pulmonary arterial vessels, which concomitantly displayed strong vasoconstriction. Moreover, pneumolysin increased permeability of HUVEC and A549 monolayers. Interestingly, immunofluorescence of endothelial cell monolayers exposed to pneumolysin showed gap formation and moderate stress fiber generation., Conclusions: Pneumolysin may play a central role for early-onset acute lung injury due to severe pneumococcal pneumonia by causing impairment of pulmonary microvascular barrier function and severe pulmonary hypertension.

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.

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.