Torsten Loop

Suppression of inflammatory cytokine production by carbon monoxide involves the JNK pathway and AP-1.

The stress-inducible protein heme oxygenase-1 provides protection against oxidative stress and modulates pro-inflammatory cytokines. As the sepsis syndrome results from the release of pro-inflammatory mediators, we postulated that heme oxygenase-1 and its enzymatic product CO would protect against lethality in a murine model of sepsis. Mice treated with a lethal dose of lipopolysaccharide (LPS) and subsequently exposed to inhaled CO had significantly better survival and lower serum interleukin (IL)-6 and IL-1β levels than their untreated counterparts. In vitro, mouse macrophages exposed to LPS and CO had significantly attenuated IL-6 production; this effect was concentration-dependent and occurred at a transcriptional level. The same effect was seen with increased endogenous CO production through overexpression of heme oxygenase-1. Mutation within the AP-1-binding site in the IL-6 promoter diminished the effect of CO on promoter activity, and treatment of macrophages with CO decreased AP-1 binding in an electrophoretic mobility shift assay. Electrophoretic mobility supershift assay indicated that the JunB, JunD, and c-Fos components of AP-1 were particularly affected. Upstream of AP-1, CO decreased JNK phosphorylation in murine macrophages and lung endothelial cells. Mice deficient in the JNK pathway had decreased serum levels of IL-6 and IL-1β in response to LPS compared with control mice, and no effect of CO on these cytokine levels was seen in Jnk1 or Jnk2 genedeleted mice. In summary, these results suggest that CO provides protection in a murine model of sepsis through modulation of inflammatory cytokine production. For the first time, the effect of CO is shown to be mediated via the JNK signaling pathway and the transcription factor AP-1.

Extracorporeal membrane oxygenation: a ten-year experience

BACKGROUND Extracorporeal membrane oxygenation (ECMO) is a supportive therapy used for severe acute respiratory distress syndrome (ARDS). We present outcome, clinical parameters, and complications in a cohort of 245 ARDS patients of whom 62 were treated with ECMO. METHODS Data of all ARDS patients were prospectively collected between 1991 and 1999. Outcome and clinical parameters of patients treated with and without ECMO were evaluated. RESULTS One hundred thirty-eight patients were referred from other hospitals, 107 were primarily located in our hospital. About one fourth of these patients were treated with ECMO. The survival rate was 55% in ECMO patients and 61% in non-ECMO patients. CONCLUSIONS ECMO is a therapeutic option for patients with severe ARDS, likely to increase survival. However, a randomized controlled study proving its benefit is still awaited. Until the development of a causal or otherwise superior therapy ECMO should be used in selected patients.

Volatile anesthetics induce caspase-dependent, mitochondria-mediated apoptosis in human T lymphocytes in vitro.

Background:Volatile anesthetics modulate lymphocyte function during surgery, and this compromises postoperative immune competence. The current work was undertaken to examine whether volatile anesthetics induce apoptosis in human T lymphocytes and what apoptotic signaling pathway might be used. Methods:Effects of sevoflurane, isoflurane, and desflurane were studied in primary human CD3+ T lymphocytes and Jurkat T cells in vitro. Apoptosis and mitochondrial membrane potential were assessed using flow cytometry after green fluorescent protein-annexin V and DiOC6-fluorochrome staining. Activity and proteolytic processing of caspase 3 was measured by cleaving of the fluorogenic effector caspase substrate Ac-DEVD-AMC and by anti–caspase-3 Western blotting. Release of mitochondrial cytochrome c was studied after cell fractionation using anti–cytochrome c Western blotting and enzyme-linked immunosorbent assays. Results:Sevoflurane and isoflurane induced apoptosis in human T lymphocytes in a dose-dependent manner. By contrast, desflurane did not exert any proapoptotic effects. The apoptotic signaling pathway used by sevoflurane involved disruption of the mitochondrial membrane potential and release of cytochrome c from mitochondria to the cytosol. In addition, the authors observed a proteolytic cleavage of the inactive p32 procaspase 3 to the active p17 fragment, increased caspase-3–like activity, and cleavage of the caspase-3 substrate poly-ADP-ribose-polymerase. Sevoflurane-induced apoptosis was blocked by the general caspase inhibitor Z-VAD.fmk. Death signaling was not mediated via the Fas/CD95 receptor pathway because neither anti-Fas/CD95 receptor antagonism nor FADD deficiency or caspase-8 deficiency were able to attenuate sevoflurane-mediated apoptosis. Conclusion:Sevoflurane and isoflurane induce apoptosis in T lymphocytes via increased mitochondrial membrane permeability and caspase-3 activation, but independently of death receptor signaling.

Heme oxygenase-1 induction by the clinically used anesthetic isoflurane protects rat livers from ischemia/reperfusion injury.

Objective:It was the aim of this study to characterize the influence of isoflurane-induced heme oxygenase-1 (HO-1) expression on hepatocellular integrity after ischemia and reperfusion. Summary Background Data:Abundant experimental data characterize HO-1 as one of the most powerful inducible enzymes that contribute to the protection of the liver and other organs after harmful stimuli. Therapeutic strategies aimed at utilizing the protective effects of HO-1 are hampered by the fact that most pharmacological inducers of this enzyme perturb organ function by themselves and are not available for use in patients because of their toxicity and undesirable or unknown side effects. Methods:Rats were pretreated with isoflurane before induction of partial hepatic ischemia (1 hour) and reperfusion (1 hour). At the end of each experiment, blood and liver tissue were obtained for molecular biologic, histologic, and immunohistochemical analyses. Results:Isoflurane pretreatment increased hepatic HO-1 mRNA, HO-1 protein, HO enzyme activity, and decreased plasma levels of AST, ALT, and α-GST. Histologic analysis of livers obtained from isoflurane-pretreated rats showed a reduction of necrotic areas, particularly in the perivenular region, the predominant site of isoflurane-induced HO-1 expression. In addition, sinusoidal congestion that could otherwise be observed after ischemia/reperfusion was inhibited by the anesthetic. Furthermore, isoflurane augmented hepatic microvascular blood flow and lowered the malondialdehyde content within the liver compared with control animals. Administration of tin protoporphyrin IX inhibited HO activity and abolished the isoflurane-induced protective effects. Conclusions:This study provides first evidence that pretreatment with the nontoxic and clinically approved anesthetic isoflurane induces hepatic HO-1 expression, and thereby protects rat livers from ischemia/reperfusion injury.

Inhaled hydrogen sulfide protects against ventilator-induced lung injury.

Background:Mechanical ventilation still causes an unacceptably high rate of morbidity and mortality because of ventilator-induced lung injury (VILI). Therefore, new therapeutic strategies are needed to treat VILI. Hydrogen sulfide can induce hypothermia and suspended animation-like states in mice. Hydrogen sulfide can also confer antiinflammatory and antiapoptotic effects. This study investigates the organ-protective effects of inhaled hydrogen sulfide during mechanical ventilation. Methods:Mice were ventilated with a tidal volume of 12 ml/kg body weight for 6 h with synthetic air in the absence or presence of hydrogen sulfide (80 parts per million) and, in a second series, at either mild hypothermia or normothermia. Staining of lung sections determined the degree of lung damage by VILI score and apoptotic cells. Bronchoalveolar lavage fluid was analyzed for the cytokines interleukin-1&bgr; and macrophage inflammatory protein-1&bgr; and for neutrophil accumulation. Heme oxygenase-1 and heat shock protein 70 expression were assessed in the lung tissue by Western immunoblot analysis. Results:Mechanical ventilation at both hypothermia and normothermia led to a profound development of VILI, characterized by pulmonary edema, increased apoptosis, cytokine release, neutrophil recruitment, and up-regulation of the stress proteins such as heme oxygenase-1 and heat shock protein 70. In contrast, the application of hydrogen sulfide during ventilation at either mild hypothermia or normothermia prevented edema formation, apoptosis, proinflammatory cytokine production, neutrophil accumulation, and inhibited heme oxygenase-1 expression. Conclusions:Inhalation of hydrogen sulfide during mechanical ventilation protects against VILI by the inhibition of inflammatory and apoptotic responses. Hydrogen sulfide confers lung protection independently of its ability to induce mild hypothermia during ventilation.

Thiopental Inhibits the Activation of Nuclear Factor κB

Background Thiopental is frequently used for the treatment of intracranial hypertension after severe head injury. Its long-term administration increases the incidence of nosocomial infections, which contributes to the high mortality rate of these patients. However, the mechanism of its immunosuppressing effect remains unknown. Methods The effect of thiopental (200–1000 &mgr;g/ml) on the activation of the nuclear transcription factor &kgr;B (NF-&kgr;B; electrophoretic mobility shift assays), on NF-&kgr;B–driven reporter gene activity (transient transfection assays), on the expression of NF-&kgr;B target genes (enzyme-linked immunoassays), on T-cell activation (flow cytometric analyses of CD69 expression), and on the content of the NF-&kgr;B inhibitor I&kgr;B-&agr; (Western blotting) was studied in human T lymphocytes in vitro. Results Thiopental inhibited the activation of the transcription factor NF-&kgr;B but did not alter the activity of the cyclic adenosine monophosphate response element binding protein. Other barbiturates (methohexital), anesthetics (etomidate, propofol, ketamine), or opioids (fentanyl, morphine) did not affect NF-&kgr;B activation. Thiopental-mediated suppression of NF-&kgr;B could be observed in Jurkat cells and in primary CD3+ lymphocytes from healthy volunteers, was time- and concentration-dependent, occurred at concentrations that are clinically achieved, and persisted for hours after the incubation. It was associated with an inhibition of NF-&kgr;B–driven reporter gene activity, of the expression of interleukin-2, -6, and -8, and interferon &ggr;, and of the activation of CD3+ lymphocytes. Suppression of NF-&kgr;B appeared to involve reduced degradation of I&kgr;B-&agr;. Conclusion The results demonstrate that thiopental inhibits the activation of NF-&kgr;B and may thus provide a molecular mechanism for some of the immunosuppressing effects associated with thiopental therapy.

Pulsatile Pulmonary Perfusion During Cardiopulmonary Bypass Reduces the Pulmonary Inflammatory Response

BACKGROUND Pulmonary dysfunction presumably linked to an inflammatory response is frequent after cardiac operations using cardiopulmonary bypass (CPB) and pulmonary hypoperfusion. We previously demonstrated that active perfusion of the lungs during CPB reduces ischemic lung injury. We now hypothesized that avoiding ischemia of the lungs during CPB by active pulmonary perfusion would decrease pulmonary inflammatory response. METHODS Pigs were randomized to a control group with CPB for 120 minutes, followed by 120 minutes of postbypass reperfusion, or to the study groups where animals underwent active pulmonary perfusion with pulsatile or nonpulsatile perfusion during CPB (n = 7 in each group). Activation of transcription factor activity (nuclear factor [NF]-kappaB and activating protein [AP]-1) was determined by electrophoretic mobility shift assay. Levels of proinflammatory protein expression (interleukin [IL]-1, IL-6, and tumor necrosis factor [TNF]-alpha) were quantified by enzyme-linked immunoabsorbent assay. Caspase-3 activity was measured using a fluorogenic assay. RESULTS The activation of transcription factor AP-1 and NF-kappaB was reduced in the pulsatile pulmonary perfusion group. The caspase-3 activity and the expression of IL-1, IL-6, and TNF-alpha revealed a significant decrease in the pulsatile and nonpulsatile pulmonary perfusion groups. Animals of the pulsatile pulmonary perfusion group showed significantly reduced IL-6 expression and caspase-3 activity compared with the nonpulsatile pulmonary perfusion group. CONCLUSIONS Active pulmonary perfusion reduces the inflammatory response and apoptosis in the lungs observed during conventional CPB. This effect is greatest when pulmonary perfusion is performed with pulsatility. The reduction in cytokine expression by pulsatile pulmonary perfusion might be mediated by AP-1 and NF-kappaB.

Carbon Monoxide Inhalation Reduces Pulmonary Inflammatory Response during Cardiopulmonary Bypass in Pigs

Background:Cardiopulmonary bypass (CPB) is associated with pulmonary inflammation and dysfunction. This may lead to acute lung injury and acute respiratory distress syndrome with increased morbidity and mortality. The authors hypothesized that inhaled carbon monoxide before initiation of CPB would reduce inflammatory response in the lungs. Methods:In a porcine model, a beating-heart CPB was used. The animals were either randomized to a control group, to standard CPB, or to CPB plus carbon monoxide. In the latter group, lungs were ventilated with 250 ppm inhaled carbon monoxide in addition to standard ventilation before CPB. Lung tissue samples were obtained at various time points, and pulmonary cytokine levels were determined. Results:Hemodynamic parameters were largely unaffected by CPB or carbon monoxide inhalation. There were no significant differences in cytokine expression in mononuclear cells between the groups throughout the experimental time course. Compared with standard CPB animals, carbon monoxide significantly suppresses tumor necrosis factor-&agr; and interleukin-1&bgr; levels (P < 0.05) and induced the antiinflammatory cytokine interleukin 10 (P < 0.001). Carbon monoxide inhalation modulates effector caspase activity in lung tissue during CPB. Conclusions:The results demonstrate that inhaled carbon monoxide significantly reduces CPB-induced inflammation via suppression of tumor necrosis factor &agr;, and interleukin-1&bgr; expression and elevation of interleukin 10. Apoptosis induced by CPB was associated with caspase-3 activation and was significantly attenuated by carbon monoxide treatment. Based on the observations of this study, inhaled carbon monoxide could represent a potential new therapeutic modality for counteracting CPB-induced lung injury.

Thiopental Inhibits Tumor Necrosis Factor α–induced Activation of Nuclear Factor κB through Suppression of IκB Kinase Activity

Background Thiopental is frequently used for the treatment of intracranial hypertension after severe head injury and is associated with immunosuppressive effects. The authors have recently reported that thiopental inhibits activation of nuclear factor (NF) &kgr;B, a transcription factor implicated in the expression of many inflammatory genes. Thus, it was the aim of the current study to examine the molecular mechanism of this inhibitory effect. Methods The authors tested &ggr;-aminobutyric acid (GABA), the GABAA antagonist bicuculline, and the GABAB antagonist dichlorophenyl-methyl-amino-propyl-diethoxymethyl-phosphinic acid (CGP 52432) in combination with thiopental for their influence on the activation of NF-&kgr;B. In addition, they investigated the direct effect of thiopental on activated NF-&kgr;B DNA binding activity. These experiments were conducted in Jurkat T lymphocytes using electrophoretic mobility shift assays. The presence of the phosphorylated and dephosphorylated NF-&kgr;B inhibitor I&kgr;B&agr; (Western blotting) and I&kgr;B kinase activity were studied in Jurkat T cells and human CD3+ T lymphocytes. In addition, the authors tested the effect of the structural barbiturate analog pairs thiopental–pentobarbital and thiamylal–secobarbital and of thiopental in combination with the thio-group containing chemical dithiothreitol on the activation of NF-&kgr;B. Results GABA did not inhibit NF-&kgr;B activation, and the GABAA and GABAB antagonists bicuculline and CGP did not diminish the thiopental-mediated inhibitory effect on NF-&kgr;B activation. Thiopental did not inhibit activated NF-&kgr;B directly in a cell-free system. The phosphorylation of I&kgr;B&agr; was prevented after incubation with 1,000 &mgr;g/ml thiopental. The same concentration of thiopental also inhibited I&kgr;B kinase activity in tumor necrosis factor–stimulated Jurkat T cells and human CD3+ T lymphocytes (60% suppression, P < 0.05 vs. tumor necrosis factor &agr; alone). Thiobarbiturates (4 × 10−3 m) inhibited NF-&kgr;B activity, whereas equimolar concentrations of the structural oxyanalogs did not. Preincubation of thiopental with dithiothreitol diminished the inhibitory effect. Conclusion Thiopental-mediated inhibition of NF-&kgr;B activation is due to the suppression of I&kgr;B kinase activity and depends at least in part on the thio-group of the barbiturate molecule.

Mechanism of hepatic heme oxygenase-1 induction by isoflurane

Background:The heme oxygenase pathway represents a major cell and organ protective system in the liver. The authors recently showed that isoflurane and sevoflurane up-regulate the inducible isoform heme oxygenase 1 (HO-1). Because the activating cascade remained unclear, it was the aim of this study to identify the underlying mechanism of this effect. Methods:Rats were anesthetized with pentobarbital intravenously or with isoflurane per inhalation (2.3 vol%). Kupffer cell function was inhibited by dexamethasone or gadolinium chloride. Nitric oxide synthases were inhibited by either N&ohgr;-nitro-l-arginine methyl ester or S-methyl thiourea. N-Acetyl-cysteine served as an antioxidant, and diethyldithiocarbamate served as an inhibitor of cytochrome P450 2E1. Protein kinase C and phospholipase A2 were inhibited by chelerythrine or quinacrine, respectively. HO-1 was analyzed in liver tissue by Northern blot, Western blot, immunostaining, and enzymatic activity assay. Results:In contrast to pentobarbital, isoflurane induced HO-1 after 4–6 h in hepatocytes in the pericentral region of the liver. The induction was prevented in the presence of dexamethasone (P < 0.05) and gadolinium chloride (P < 0.05). Inhibition of nitric oxide synthases or reactive oxygen intermediates did not affect isoflurane-mediated HO-1 up-regulation. In contrast, chelerythrine (P < 0.05) and quinacrine (P < 0.05) resulted in a blockade of HO-1 induction. Conclusion:The up-regulation of HO-1 by isoflurane in the liver is restricted to parenchymal cells and depends on Kupffer cell function. The induction is independent of nitric oxide or reactive oxygen species but does involve protein kinase C and phospholipase A2.