Ulrike Uhlig

The Effects of Different Ventilatory Settings on Pulmonary and Systemic Inflammatory Responses During Major Surgery

Mechanical ventilation with high tidal volumes (VT) and zero or low positive end-expiratory pressure increased mediator release to inflammatory stimuli or acute lung injury. We studied whether mechanical ventilation modifies the inflammatory responses during major thoracic or abdominal surgery. Sixty-four patients undergoing elective thoracotomy (n = 34) or laparotomy (n = 30) were randomized to receive either mechanical ventilation with VT = 12 or 15 mL/kg ideal body weight, respectively, and zero end-expiratory pressure, or VT = 6 mL/kg ideal body weight with positive end-expiratory pressure of 10 cm H2O. In 62 patients who completed the study, arterial oxygena- tion was not different between groups. Tumor necrosis factor, interleukin (IL)-1, IL-6, IL-8, IL-10, and IL-12 were determined by cytometric bead array in plasma after 0, 1, 2, and 3 h and in tracheal aspirates after 3 h of mechanical ventilation. Data were log-transformed and analyzed using parametric or nonparametric tests, as indicated. All plasma mediators increased more during abdominal than during thoracic surgery, although the differences were small. However, neither time course nor concentrations of pulmonary or systemic mediators differed between the two ventilatory settings. Our data suggest that the ventilatory settings we studied do not affect inflammatory reactions during major surgery within 3 h.

Models and mechanisms of acute lung injury caused by direct insults

Acute lung injury (ALI) and its more severe form acute respiratory distress syndrome (ARDS) are life-threatening diseases that are characterized by acute onset, pulmonary inflammation, oedema due to increased vascular permeability and severe hypoxemia. Clinically, ARDS can be divided into ARDS due to direct causes such as pneumonia, aspiration or injurious ventilation, and due to extrapulmonary indirect causes such as sepsis, severe burns or pancreatitis. In order to identify potential therapeutic targets, we asked here whether common molecular mechanisms can be identified that are relevant in different models of the direct form of ALI/ARDS. To this end, we reviewed three widely used models: (a) one based on a biological insult, i.e. instillation of bacterial endotoxins; (b) one based on a chemical insult, i.e. instillation of acid; and (c) one based on a mechanical insult, i.e. injurious ventilation. Studies were included only if the mediator or mechanism of interest was studied in at least two of the three animal models listed above. As endpoints, we selected neutrophil sequestration, permeability, hypoxemia (physiological dysfunction) and survival. Our analysis showed that most studies have focused on mechanisms of pulmonary neutrophil sequestration and models with moderate forms of oedema. The underlying mechanisms that involve canonical inflammatory pathways such as MAP kinases, CXCR2 chemokines, PAF, leukotrienes, adhesions molecules (CD18, ICAM-1) and elastase have been defined relatively well. Further mechanisms including TNF, DARC, HMGB1, PARP, GADD45 and collagenase are under investigation. Such mechanisms that are shared between the three ALI models may represent viable therapeutic targets. However, only few studies have linked these pathways to hypoxemia, the most important clinical aspect of ALI/ARDS. Since moderate oedema does not necessarily lead to hypoxemia, we suggest that the clinical relevance of experimental studies can be further improved by putting greater emphasis on gas exchange.

Pulmonary cytokine responses during mechanical ventilation of noninjured lungs with and without end-expiratory pressure.

BACKGROUND: Positive end-expiratory pressure (PEEP) during mechanical ventilation may impose different degrees of stress on healthy lungs. On the assumption that stress is reflected by cytokine production, we performed a translational study investigating the effect of PEEP on bronchoalveolar and systemic mediator levels in isolated perfused mouse lungs (IPL) and in patients with healthy lungs. METHODS: (Part I) IPL were ventilated with end-expiratory pressures of 0, 3, 6, or 10 cm H2O and end-inspiratory pressure (EIP) levels of 10 or 25 cm H2O. Interleukin (IL)-6 and macrophage inflammatory protein-2 concentrations in the venous effluate were monitored. (Part II) Patients (nonsmokers) scheduled for elective otorhinolaryngology surgery (duration >90 min) were randomized to receive either ventilation with zero end-expiratory pressure or PEEP (10 cm H2O). Mediators in bronchoalveolar lavage, nuclear factor &kgr;B, (NF-&kgr;B)-activation in alveolar macrophages and circulating systemic mediators were monitored. Control patients underwent bronchoalveolar lavage after intubation. RESULTS: In the IPL, mediator concentrations increased with increasing end-expiratory pressure at an EIP of 10 cm H2O, but decreased at 25 cm H2O EIP. In patients, bronchoalveolar IL-6, monocyte chemoattractant protein-1, and granulocyte monocyte-colony stimulating factor were increased by ventilation regardless of the PEEP level. IL-6 and IL-8 levels were moderately increased by PEEP but not zero end-expiratory pressure. Nuclear factor &kgr;B DNA binding activity in alveolar macrophages and systemic mediator levels did not change. CONCLUSIONS: On the basis of the premise that cytokine levels may indicate mechanical stress, our findings indicate that even low tidal volume ventilation causes some stress. PEEP is beneficial at high inspiratory pressure, but imposes moderate stress at low inspiratory pressure.

Ventilation-Induced Lung Injury

Mechanical ventilation (MV) is, by definition, the application of external forces to the lungs. Depending on their magnitude, these forces can cause a continuum of pathophysiological alterations ranging from the stimulation of inflammation to the disruption of cell-cell contacts and cell membranes. These side effects of MV are particularly relevant for patients with inhomogeneously injured lungs such as in acute lung injury (ALI). These patients require supraphysiological ventilation pressures to guarantee even the most modest gas exchange. In this situation, ventilation causes additional strain by overdistension of the yet non-injured region, and additional stress that forms because of the interdependence between intact and atelectatic areas. Cells are equipped with elaborate mechanotransduction machineries that respond to strain and stress by the activation of inflammation and repair mechanisms. Inflammation is the fundamental response of the host to external assaults, be they of mechanical or of microbial origin and can, if excessive, injure the parenchymal tissue leading to ALI. Here, we will discuss the forces generated by MV and how they may injure the lungs mechanically and through inflammation. We will give an overview of the mechanotransduction and how it leads to inflammation and review studies demonstrating that ventilator-induced lung injury can be prevented by blocking pathways of mechanotransduction or inflammation.

Ceramide alters endothelial cell permeability by a nonapoptotic mechanism

1 Ceramide is a lipid second messenger that was recently identified as mediator of pulmonary edema in vivo. Here, we investigated the effect of ceramide on the permeability of confluent endothelial cell monolayers. 2 In monolayers of bovine pulmonary artery and human microvascular pulmonary endothelial cells, incubation with C6‐ceramide for 3 h elevated permeability in a concentration‐dependent manner, whereas dihydroceramide was without effect. 3 After 3 h of incubation with ceramide, we found no signs of necrosis (release of lactate dehydrogenase, loss of thiazylyl blue reduction) or apoptosis (ssDNA, caspase‐8 activity). 4 The increased endothelial permeability in response to ceramide was attenuated by the Ser/Thr protein kinase inhibitors K252a, K252b and H‐7, as well as by the phosphatidylinositol‐specific phospholipase C inhibitor L108. Since in some systems sphingosine‐1‐phosphate (S1P) acts antagonistic to ceramide, the effect of S1P was studied. S1P transiently increased endothelial cell resistance, whether it was given together with ceramide or 90 min thereafter. 5 These data provide a novel example of the antagonism between S1P and ceramide. Our findings further suggest that ceramide alters vascular permeability by activation of pathways dependent on unidentified phospholipase C and Ser/Thr kinase isoenzymes.

IKK NBD peptide inhibits LPS induced pulmonary inflammation and alters sphingolipid metabolism in a murine model.

Airway epithelial NF-κB is a key regulator of host defence in bacterial infections and has recently evolved as a target for therapeutical approaches. Evidence is accumulating that ceramide, generated by acid sphingomyelinase (aSMase), and sphingosine-1-phosphate (S1-P) are important mediators in host defence as well as in pathologic processes of acute lung injury. Little is known about the regulatory mechanisms of pulmonary sphingolipid metabolism in bacterial infections of the lung. The objective of this study was to evaluate the influence of NF-κB on sphingolipid metabolism in Pseudomonas aeruginosa LPS-induced pulmonary inflammation. In a murine acute lung injury model with intranasal Pseudomonas aeruginosa LPS we investigated TNF-α, KC (murine IL-8), IL-6, MCP-1 and neutrophilic infiltration next to aSMase activity and ceramide and S1-P lung tissue concentrations. Airway epithelial NF-κB was inhibited by topically applied IKK NBD, a cell penetrating NEMO binding peptide. This treatment resulted in significantly reduced inflammation and suppression of aSMase activity along with decreased ceramide and S1-P tissue concentrations down to levels observed in healthy animals. In conclusion our results confirm that changes in sphingolipid metabolim due to Pseudomonas aeruginosa LPS inhalation are regulated by NF-κB translocation. This confirms the critical role of airway epithelial NF-κB pathway for the inflammatory response to bacterial pathogens and underlines the impact of sphingolipids in inflammatory host defence mechanisms.

Pulmonary responses to overventilation in late multiple organ failure

Background:Patients with multiple organ failure (MOF) require mechanical ventilation for several days. The enormous significance of the ventilation strategy for the outcome of these patients is well appreciated. However, most studies have focused on the onset and the early phase of MOF. It was the aim of the current study to investigate the effect of ventilation in the course of MOF. Methods:Using a model where mice develop MOF 7–14 days after intraperitoneal injection of zymosan, the authors analyzed lung functions, signaling pathways, and mediator release in response to protective ventilation (end-expiratory pressure −3 cm H2O; end-inspiratory pressure −10 cm H2O) and overventilation (−22.5 cm H2O) in isolated lungs ex vivo. Results:On day 7, pulmonary compliance, pulmonary resistance, and tidal volume were normal, but vascular resistance was elevated compared with untreated animals. During ex vivo ventilation, these lungs showed enhanced nuclear factor-&kgr;B activation, Akt kinase phosphorylation, and cytokine release, and this was further aggravated by overventilation. After 14 days, zymosan-treated animals were characterized by pulmonary hypertension, reduced tidal volume, elevated pulmonary resistance, and increased mediator production. However, in these lungs, neither nuclear factor-&kgr;B activation nor cytokine production where enhanced by overventilation. Conclusions:The zymosan model is characterized by pulmonary inflammation, diminished lung functions, and chronic hypertension. Mechanical ventilation with high distending pressures further augmented cytokine production in this chronic model of MOF only if it significantly augmented tidal volume. The authors speculate that these findings may be explained on the basis of different degrees of lung stretch.

Interplay between nuclear factor erythroid 2-related factor 2 and amphiregulin during mechanical ventilation.

Mechanical ventilation (MV) elicits complex and clinically relevant cellular responses in the lungs. The current study was designed to define the role of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), a major regulator of the cellular antioxidant defense system, in the pulmonary response to MV. Nrf2 activity was quantified in ventilated isolated perfused mouse lungs (IPL). Regulation of amphiregulin (AREG) was investigated in BEAS-2B cells with inactivated Nrf2 or Keap1, the inhibitor of Nrf2, using a luciferase vector with AREG promoter. AREG-dependent Nrf2 activity was examined in BEAS-2B cells, murine precision-cut lung slices (PCLS), and IPL. Finally, Nrf2 knockout and wild-type mice were ventilated to investigate the interplay between Nrf2 and AREG during MV in vivo. Lung functions and inflammatory parameters were measured. Nrf2 was activated in a ventilation-dependent manner. The knockdown of Nrf2 and Keap1 via short hairpin RNA in BEAS-2B cells and an EMSA with lung tissue revealed that AREG is regulated by Nrf2. Conversely, AREG application induced a significant Nrf2 activation in BEAS-2B cells, PCLS, and IPL. The signal transduction of ventilation-induced Nrf2 activation was shown to be p38 MAP kinase-dependent. In vivo ventilation experiments indicated that AREG is regulated by Nrf2 during MV. We conclude that Areg expression is regulated by Nrf2. During high-pressure ventilation, Nrf2 becomes activated and induces AREG, leading to a positive feedback loop between Nrf2 and AREG, which involves the p38 MAPK and results in the expression of cytoprotective genes.

Inflammatory Mediators in Tracheal Aspirates of Preterm Infants Participating in a Randomized Trial of Inhaled Nitric Oxide

Background Ventilated preterm infants frequently develop bronchopulmonary dysplasia (BPD) which is associated with elevated inflammatory mediators in their tracheal aspirates (TA). In animal models of BPD, inhaled nitric oxide (iNO) has been shown to reduce lung inflammation, but data for human preterm infants is missing. Methods Within a European multicenter trial of NO inhalation for preterm infants to prevent BPD (EUNO), TA was collected to determine the effects of iNO on pulmonary inflammation. TA was collected from 43 premature infants randomly assigned to receive either iNO or placebo gas (birth weight 530–1230 g, median 800 g, gestational age 24 to 28 2/7 weeks, median 26 weeks). Interleukin (IL)-1β, IL-6, IL-8, transforming growth factor (TGF)-β1, interferon γ-induced protein 10 (IP-10), macrophage inflammatory protein (MIP)-1α, acid sphingomyelinase (ASM), neuropeptide Y and leukotriene B4 were measured in serial TA samples from postnatal day 2 to 14. Furthermore, TA levels of nitrotyrosine and nitrite were determined under iNO therapy. Results The TA levels of IP-10, IL-6, IL-8, MIP-1α, IL-1β, ASM and albumin increased with advancing postnatal age in critically ill preterm infants, whereas nitrotyrosine TA levels declined in both, iNO-treated and placebo-treated infants. The iNO treatment generally increased nitrite TA levels, whereas nitrotyrosine TA levels were not affected by iNO treatment. Furthermore, iNO treatment transiently reduced early inflammatory and fibrotic markers associated with BPD development including TGF-β1, IP-10 and IL-8, but induced a delayed increase of ASM TA levels. Conclusion Treatment with iNO may have played a role in reducing several inflammatory and fibrotic mediators in TA of preterm infants compared to placebo-treated infants. However, survival without BPD was not affected in the main EUNO trial. Trial registration NCT00551642

Inflammatory Mediators in Tracheal Aspirates of Preterm Infants Participating in a Randomized Trial of Permissive Hypercapnia

Background Ventilator-induced lung injury is considered to be a main factor in the pathogenesis of bronchopulmonary dysplasia (BPD). Optimizing ventilator strategies may reduce respiratory morbidities in preterm infants. Permissive hypercapnia has been suggested to attenuate lung injury. We aimed to determine if a higher PCO2 target range results in less lung injury compared to the control target range and possibly reduces pro-inflammatory cytokines and acid sphingomyelinase (ASM) in tracheal aspirates (TA), which has not been addressed before. Methods During a multicenter trial of permissive hypercapnia in extremely low birthweight infants (PHELBI), preterm infants (birthweight 400–1,000 g, gestational age 23 0/7–28 6/7 weeks) requiring mechanical ventilation within 24 h of birth were randomly assigned to a high PCO2 target or a control group. The high target group aimed at PCO2 values of 55–65, 60–70, and 65–75 mmHg and the control group at PCO2 values of 40–50, 45–55 and 50–60 mmHg on postnatal days 1–3, 4–6, and 7–14, respectively. TA was analyzed for pro-inflammatory cytokines from postnatal day 2–21. BPD was determined at a postmenstrual age of 36 weeks ± 2 days. Main findings Levels of inflammatory cytokines and ASM were similar in both groups: interleukin (IL)-6 (p = 0.14), IL-8 (p = 0.43), IL-10 (p = 0.24), IL-1β (p = 0.11), macrophage inflammatory protein 1α (p = 0.44), albumin (p = 0.41), neuropeptide Y (p = 0.52), leukotriene B4 (p = 0.11), transforming growth factor-β1 (p = 0.68), nitrite (p = 0.15), and ASM (p = 0.94). Furthermore, most inflammatory mediators were strongly affected by the age of the infants and increased from postnatal day 2 to 21. BPD or death was observed in 14 out of 62 infants, who were distributed evenly between both groups. Conclusion The results suggest that high PCO2 target levels did not result in lower pulmonary inflammatory activity and thus reflect clinical results. This indicates that high PCO2 target ranges are not effective in reducing ventilator-induced lung injury in preterm infants, as compared to control targets. Trial registration ISRCTN56143743.