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Role of Antioxidative Defence in Activation Induced Cell Death
to improving mouse survivals after post-burn infection.
403
doi: 10.1016/j.freeradbiomed.2010.10.410
Role of Antioxidative Defence in Activation Induced Cell Death
401 Attenuation of the Inflammatory Response in a Mouse Colitis Model by Antioxidant Luteolin: Key Role of Aglycone in Inactivation of Macrophages Cocultured with Intestinal Epithelial Cells in vitro Yosuke Nishitani1, and Masashi Mizuno1 1 Kobe University Luteolin is a well-known flavonoid that exerts anti-inflammatory properties. In this study, we investigated whether luteolin exerted gut anti-inflammatory effects. In DSS-induced colitis mouse model, luteolin (20 and 50 mg/kg) significantly ameliorated shortening of colon length and histological score of the colon. Immunohistochemical analysis showed that luteolin significantly + inhibited infiltration of macrophage and IFN-γ producing CD4 T cells into colonic mucosa. In the cellular level assessment using a co-culture system consisting of Caco-2 and RAW264.7 cells stimulated by LPS, the addition of luteolin (100 μM) to the apical side suppressed IL-8 mRNA expression in Caco-2. TNF-α production, which is essential for the up-regulation of IL-8 mRNA expression, from RAW264.7 was also suppressed by inhibition of NF-κB nuclear translocation. HPLC and subsequent cellular assay revealed that aglycone of luteolin was present in the basolateral supernatant of this system at a concentration enough to suppress activation of RAW264.7. These results suggest that aglycone of luteolin, which was absorbed and released from Caco-2 epithelium without metabolism, might inhibit NF-κB activation in RAW264.7, followed by reduction of TNF-α mRNA expression and production, resulting in down-regulation of IL-8 mRNA expression in Caco-2.
Daniel Roeth1, Marcin M. Kaminski1, Peter H. Krammer1, and Karsten Gulow1 1 German Cancer Research Center DKFZ Termination of an immune response requires elimination of activated T lymphocytes by activation induced T cell death (AICD). In AICD, CD95L (APO-1L, FasL) triggers apoptosis of CD95 positive activated T cells. A hydrogen peroxide signal combined with a simultaneous calcium influx into the cytosol is the minimal requirement for induction of CD95L expression. Either signal alone is insufficient. Recently we have shown that the proximal T cell receptor (TCR) machinery including ZAP70, LAT, SLP76, PLC gamma and PKC theta is crucial for the production of reactive oxygen species (ROS). After activation a subpopulation of PKC theta translocates to the mitochondria. At the mitochondrion we indentified complex I of the electron transport chain as the source of activation induced ROS. Since changes in the redox equilibrium of a cell are not only controlled in the phase of ROS production but are also influenced by anti-oxidative defence mechanisms we investigated the role of anti-oxidative enzymes in the process of AICD. Manganese superoxide dismutase (MnSOD) an enzyme of the mitochondrial matrix is upregulated upon TCR stimulation indicating a role in the regulation of ROS production, activation of transcription factors (e.g. NF-κB and AP-1), gene expression (e.g. CD95L) and finally AICD. Thus, this work provides new insights into regulatory mechanisms of a T cell immune response and therefore, will help to better understand immune disorders caused by deregulation of oxidative signals. doi: 10.1016/j.freeradbiomed.2010.10.413
doi: 10.1016/j.freeradbiomed.2010.10.411
404
402
Differential Contribution of Apoptosis and Necrosis to Ethanolinduced Liver Injury in Mice
HMGB1 in HyperoxiaCompromised Host Ability to Clear Pseudomonas aeruginosa Pneumonia Vivek Patel1, Ravikumar Sitapara1, Ashwini Gore1, Binh Phan1, Ritu Malla1, and Lin Mantell1 1 St. John's University College of Pharmacy Mechanical ventilation with supraphysiological concentrations of oxygen (hyperoxia) is routinely used to treat patients with depressed respiratory system. However, patients on ventilators have increased incidence of inflammatory lung injury and enhanced susceptibility to bacterial infection. Pseudomonas aeruginosa is one of the most common bacteria that infect patients with ventilator-associated pneumonia. Our previous studies demonstrate that hyperoxia compromises macrophage’s ability to phagocytose bacteria and HMGB1 can directly cause macrophage dysfunction. The objective of this study was to elucidate the role of HMGB1 in pneumonia caused by Pseudomonas aeruginosa under hyperoxic conditions. Here, we show that exposure to prolonged hyperoxia (≥99.0% O2) resulted in an increased lethality in C57BL/6 mice with Pseudomonas aeruginosa pneumonia. Accompanied with the increased mortality, levels of airway HMGB1 were elevated in these animals. Treatment with neutralizing anti-HMGB1 monoclonal antibody attenuated lung injury, and reduced bacterial counts in the lung and the levels of airway HMBG1 in these mice. These results indicate that HMGB1 plays a critical role in hyperoxiacompromised host ability to clear bacteria, suggesting that HMGB1 may provide a novel target in the treatment of ventilatorassociated pneumonia. doi: 10.1016/j.freeradbiomed.2010.10.412
Sanjoy Roychowdhury1, Palash Mandal1, Ariel E Feldstein2, Megan R. McMullen1, John Pollard3, and Laura E. Nagy1,4 124 3 Cleveland Clinic Foundation Vertex Pharmaceuticals (Europe) Abingdon, UK Apoptosis and necrosis are both associated with ethanol-induced injury and inflammation in the liver. However, it is not clear whether ethanol-induced necrosis is dependent or independent of apoptosis. We hypothesized that if ethanol-induced hepatic cell death is solely a consequence of initiation of apoptotic cascades, then prevention of apoptosis should attenuate ethanol-induced hepatic inflammation and liver injury. Making use of the pancaspase inhibitor VTX166 and mice deficient in Bid, a pivotal proapoptotic protein, we evaluated the contribution of apoptotic cell death in ethanol-induced hepatic inflammation, oxidative stress and liver injury. Female C57BL/6 or Bid-deficient mice were allowed free access to an ethanol-containing diet in a chronic model (25d, 32% of calories). Chronic ethanol feeding induced TUNEL and CK18 positive cells, as well as expression of the necrosis marker, receptor interacting protein kinase 3 (RIPK3), in mouse liver. Bid-deficiency, as well as treatment of wild type mice with VTX166, decreased ethanol-induced TUNEL and CK18 positive cells in the liver but did not attenuate ethanol-induced hepatic expression of RIPK3. Inhibition of apoptosis also failed to prevent ethanol-induced hepatic inflammation, accumulation of the lipid peroxidation marker 4-HNE or liver injury, assessed by ALT and AST in the circulation. In summary, ethanol feeding simultaneously induced Bid-dependent apoptotic pathways and RIPK3-mediated programmed necrosis in hepatocytes. Taken together these data suggest that activation of apoptotic pathways during ethanol exposure is a marker of hepatocellular injury but not a mediator of cytotoxicity contributing to liver damage as
SFRBM/SFRRI 2010
S147
403
doi: 10.1016/j.freeradbiomed.2010.10.410
Role of Antioxidative Defence in Activation Induced Cell Death
401 Attenuation of the Inflammatory Response in a Mouse Colitis Model by Antioxidant Luteolin: Key Role of Aglycone in Inactivation of Macrophages Cocultured with Intestinal Epithelial Cells in vitro Yosuke Nishitani1, and Masashi Mizuno1 1 Kobe University Luteolin is a well-known flavonoid that exerts anti-inflammatory properties. In this study, we investigated whether luteolin exerted gut anti-inflammatory effects. In DSS-induced colitis mouse model, luteolin (20 and 50 mg/kg) significantly ameliorated shortening of colon length and histological score of the colon. Immunohistochemical analysis showed that luteolin significantly + inhibited infiltration of macrophage and IFN-γ producing CD4 T cells into colonic mucosa. In the cellular level assessment using a co-culture system consisting of Caco-2 and RAW264.7 cells stimulated by LPS, the addition of luteolin (100 μM) to the apical side suppressed IL-8 mRNA expression in Caco-2. TNF-α production, which is essential for the up-regulation of IL-8 mRNA expression, from RAW264.7 was also suppressed by inhibition of NF-κB nuclear translocation. HPLC and subsequent cellular assay revealed that aglycone of luteolin was present in the basolateral supernatant of this system at a concentration enough to suppress activation of RAW264.7. These results suggest that aglycone of luteolin, which was absorbed and released from Caco-2 epithelium without metabolism, might inhibit NF-κB activation in RAW264.7, followed by reduction of TNF-α mRNA expression and production, resulting in down-regulation of IL-8 mRNA expression in Caco-2.
Daniel Roeth1, Marcin M. Kaminski1, Peter H. Krammer1, and Karsten Gulow1 1 German Cancer Research Center DKFZ Termination of an immune response requires elimination of activated T lymphocytes by activation induced T cell death (AICD). In AICD, CD95L (APO-1L, FasL) triggers apoptosis of CD95 positive activated T cells. A hydrogen peroxide signal combined with a simultaneous calcium influx into the cytosol is the minimal requirement for induction of CD95L expression. Either signal alone is insufficient. Recently we have shown that the proximal T cell receptor (TCR) machinery including ZAP70, LAT, SLP76, PLC gamma and PKC theta is crucial for the production of reactive oxygen species (ROS). After activation a subpopulation of PKC theta translocates to the mitochondria. At the mitochondrion we indentified complex I of the electron transport chain as the source of activation induced ROS. Since changes in the redox equilibrium of a cell are not only controlled in the phase of ROS production but are also influenced by anti-oxidative defence mechanisms we investigated the role of anti-oxidative enzymes in the process of AICD. Manganese superoxide dismutase (MnSOD) an enzyme of the mitochondrial matrix is upregulated upon TCR stimulation indicating a role in the regulation of ROS production, activation of transcription factors (e.g. NF-κB and AP-1), gene expression (e.g. CD95L) and finally AICD. Thus, this work provides new insights into regulatory mechanisms of a T cell immune response and therefore, will help to better understand immune disorders caused by deregulation of oxidative signals. doi: 10.1016/j.freeradbiomed.2010.10.413
doi: 10.1016/j.freeradbiomed.2010.10.411
404
402
Differential Contribution of Apoptosis and Necrosis to Ethanolinduced Liver Injury in Mice
HMGB1 in HyperoxiaCompromised Host Ability to Clear Pseudomonas aeruginosa Pneumonia Vivek Patel1, Ravikumar Sitapara1, Ashwini Gore1, Binh Phan1, Ritu Malla1, and Lin Mantell1 1 St. John's University College of Pharmacy Mechanical ventilation with supraphysiological concentrations of oxygen (hyperoxia) is routinely used to treat patients with depressed respiratory system. However, patients on ventilators have increased incidence of inflammatory lung injury and enhanced susceptibility to bacterial infection. Pseudomonas aeruginosa is one of the most common bacteria that infect patients with ventilator-associated pneumonia. Our previous studies demonstrate that hyperoxia compromises macrophage’s ability to phagocytose bacteria and HMGB1 can directly cause macrophage dysfunction. The objective of this study was to elucidate the role of HMGB1 in pneumonia caused by Pseudomonas aeruginosa under hyperoxic conditions. Here, we show that exposure to prolonged hyperoxia (≥99.0% O2) resulted in an increased lethality in C57BL/6 mice with Pseudomonas aeruginosa pneumonia. Accompanied with the increased mortality, levels of airway HMGB1 were elevated in these animals. Treatment with neutralizing anti-HMGB1 monoclonal antibody attenuated lung injury, and reduced bacterial counts in the lung and the levels of airway HMBG1 in these mice. These results indicate that HMGB1 plays a critical role in hyperoxiacompromised host ability to clear bacteria, suggesting that HMGB1 may provide a novel target in the treatment of ventilatorassociated pneumonia. doi: 10.1016/j.freeradbiomed.2010.10.412
Sanjoy Roychowdhury1, Palash Mandal1, Ariel E Feldstein2, Megan R. McMullen1, John Pollard3, and Laura E. Nagy1,4 124 3 Cleveland Clinic Foundation Vertex Pharmaceuticals (Europe) Abingdon, UK Apoptosis and necrosis are both associated with ethanol-induced injury and inflammation in the liver. However, it is not clear whether ethanol-induced necrosis is dependent or independent of apoptosis. We hypothesized that if ethanol-induced hepatic cell death is solely a consequence of initiation of apoptotic cascades, then prevention of apoptosis should attenuate ethanol-induced hepatic inflammation and liver injury. Making use of the pancaspase inhibitor VTX166 and mice deficient in Bid, a pivotal proapoptotic protein, we evaluated the contribution of apoptotic cell death in ethanol-induced hepatic inflammation, oxidative stress and liver injury. Female C57BL/6 or Bid-deficient mice were allowed free access to an ethanol-containing diet in a chronic model (25d, 32% of calories). Chronic ethanol feeding induced TUNEL and CK18 positive cells, as well as expression of the necrosis marker, receptor interacting protein kinase 3 (RIPK3), in mouse liver. Bid-deficiency, as well as treatment of wild type mice with VTX166, decreased ethanol-induced TUNEL and CK18 positive cells in the liver but did not attenuate ethanol-induced hepatic expression of RIPK3. Inhibition of apoptosis also failed to prevent ethanol-induced hepatic inflammation, accumulation of the lipid peroxidation marker 4-HNE or liver injury, assessed by ALT and AST in the circulation. In summary, ethanol feeding simultaneously induced Bid-dependent apoptotic pathways and RIPK3-mediated programmed necrosis in hepatocytes. Taken together these data suggest that activation of apoptotic pathways during ethanol exposure is a marker of hepatocellular injury but not a mediator of cytotoxicity contributing to liver damage as
SFRBM/SFRRI 2010
S147