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S17. Oxygen Free Radicals, Nitric Oxide and Cytokines in Inflammation
S17. Oxygen Free Radicals, Nitric Oxide and Cytokines in Inflammation
S17-88 GASTROESOPHAGEAL REFLUX DISEASE (GERD) AS AN INFLAMMATORY DISEASE T. Yoshikawa,1 Y. Naito,1 and N. Yoshida1 1 Inflammation and Immunology, Kyoto Prefectural University of Medicine, Japan
We propose a hypothesis that an inflammatory response plays a crucial role in the pathogenesis of gastroesophageal reflux disease (GERD). Interleukin 8 (IL-8) has been shown to be proinflammatory, motogenic, mitogenic, and angiogenic and to play important roles in inflammation as well as tumor progression. Among the patients with GERD, there was a significant correlation between endoscopic esophagitis grading (LA classification) and the expression level of IL-8 mRNA determined by real-time PCR. In addition, the IL-8 mRNA expression was detected in patients suffering from heart burn without endoscopic abnormal findings. Epithelial cells, infiltrating neutrophils, and monocytes are candidates for IL-8 producing cells in the esophagus. Recently we have confirmed the IL-8 production from human esophageal epithelial cell line stimulated with tumor necrosis factor-a and interleukin 1h via activation of protein kinase C, protein tyrosine kinase, and p38 MAP kinase. Especially, bile acids in acidic condition markedly induce IL-8 expression in esophageal cells via activation of AP-1, NF-IL6, and NF-nB. Experimental rat models of reflux esophagitis have also demonstrated the marked expression of IL-8 like cytokines such as CINC-1 and CINC-2. In the present paper, we discuss the role of IL-8 associated inflammation in the course of GERD.
S17-89 DIESEL EXHAUST INDUCED PULMONARY INFLAMMATION: PROTECTIVE ROLE OF AIRWAY ANTIOXIDANTS F. J. Kelly, I. S. Mudway, S. T. Duggan, A. Blomberg, A. J. Frew, and T. Sandstro¨m School of Health and Life Sciences, King’s College, London, UK; University Hospital, Umea˚, Sweden; Southampton General Hospital, Southampton, UK
Inhalation of diesel exhaust (DE) can induce pulmonary inflammation and increased airway reactivity in healthy subjects possibly through the imposition of oxidative stress. To examine this, we exposed 25 healthy subjects to an environmentally relevant concentration of DE (PM10 100 Ag m 3, 0.6 ppm NO2 for 2-hours) and filtered air on separate occasions and monitored their antioxidant defences present at the air-lung interface. Subjects exposed to DE displayed a mild bronchconstriction and a trend toward neutrophilia in proximal airways. No depletion of ascorbate (AA), urate (UA) or reduced glutathione (GSH) was
seen at any level of the respiratory tract. Instead, an increased flux of GSH into the bronchial (p < 0.01) and nasal airways (p < 0.05) was observed. To establish whether the relatively mild inflammatory response was related to the movement of antioxidants into the airway, or simply reflected a lack of DE activity, we next examined the capacity of DE to deplete antioxidants from a synthetic model of the respiratory tract lining fluid. We found DE to have comparable oxidative activity to the transition metal rich residual oil fly ash (ROFA) particle, significantly depleting both AA and GSH. These data indicate that the antioxidant network present at the air-lung interface adapts rapidly to the oxidative burden placed upon it by exposure to DE, limiting acute inflammatory responses.
S17-90 CHRONIC INFLAMMATION IN END STAGE RENAL DISEASE: MARKERS OF OXIDATIVE STRESS AND REDOX MODIFICATION A. Iannone,1 S. Bergamini,1 E. Bellei,1 C. Rota,1 and A. Tomasi1 1 Department of Biomedical Sciences, University of Modena and Reggio Emilia, 41100 Modena, Italy
End-Stage Renal Disease (ESRD) is associated with an higher incidence cardiovascular disease due to accelerated atherosclerosis, and with clinical and/or laboratory signs of systemic inflammation. This condition has been considered to be related either, to the HD and to an increased prevalence of major cardiovascular risk factors. To test the presence of oxidative stress and redox modification in ESRD patients and to evaluate the role of HD, we performed assays in the blood at the beginning and at the end of the HD session. Patients have increased level of the inhibitor of the nitrogen monoxide (NO) synthase, the asymmetric dimethylarginine (ADMA), that could lead to a reduced NO-mediated vasodilation and contribute to the initiation and progression of atherosclerosis. The content of GSH in erythrocytes is decreased and there is the appearance in blood of malondialdehyde (MDA). The HD efficiently reduces the MDA levels and also plasma nitrites, which are high before dialysis, are reduced at the end of the HD session. During the dialysis most of the ascorbic acid and dehydroascorbic acid is removed, while there are not significant variation of atocopherol, h-carotene, lycopene and retinol.
SFRR 2004
S39
S17-91
S17-92
CHOLESTEROL OXIDATION PRODUCTS AND PRO-INFLAMMATORY EFFECTS
SIGNALING TO ERK BY THE NADPH OXIDASE OF ALVEOLAR MACROPHAGES
G. Leonarduzzi,1 A. Kadl,1 B. Sottero,1 P. Gamba,1 N. Leitinger,1 and G. Poli1 1
Department of Clinical and Biological Sciences, University of Torino, S. Luigi Gonzaga Hospital, 10043 Orbassano, Torino, Italy; Department of Vascular Biology and Thrombosis Research, University of Vienna, Austria
A considerable number of studies both in animals and humans have proved that prolonged high plasma cholesterol levels increase the risk of developing atherosclerosis but the mechanisms by which cholesterol contributes to initiation and progression of atherosclerotic lesions are still subjects of debate. Cholesterol oxidation products (oxysterols) are consistently recovered in atherosclerotic plaques and may markedly enhance chronic inflammation, fibrosis and programmed cell death. Of note, unoxidised cholesterol appears by far less reactive than oxysterols, pointing to oxidation as a crucial reaction to undergo for the sterol to exert proatherogenic effects. Support to these statements also derives from investigation we performed using U937 human promonocytic cells challenged with 7-ketocholesterol, unoxidized cholesterol or a biologically representative oxysterol mixture. By means of gene expression profiling, few genes encoding for chemokines, receptors, and adhesion molecules resulted to be over-expressed only when cells were treated with the oxysterol mixture. Such evidence was validated by real time RT-PCR and immunoenzymatic analyses. Experiments are now in progress to elucidate the intracellular pathways that oxysterols activate to exert inflammationrelated gene modulation.
S40
M. Torres1 and H. J. Forman2 1
The Saban Research Institute of Childrens Hospital Los Angeles, Department of Pediatrics, Keck School of Medicine, University of Southern California, Los Angeles, CA 90027, 2School of Natural Sciences, University of California, Merced, Merced, CA 95344, USA
Alveolar macrophages (AM) respond to various stimuli such as ADP or zymosan-activated serum (ZAS) with an increase in superoxide production due to the activation of the phagocyte NADPH oxidase, NOX2. The activation of NOX2 is multi-step process, which involves the activation of several kinases that phosphorylate components of NOX2. We previously showed that AM stimulation by ADP induces activation of NOX2 without activating ERK1/2, members of the mitogen-activated protein kinase family and AKT/PKB. Interestingly, ZAS did stimulate ERK and AKT but H2O2, the product of activation of NOX2 was necessary for activation of these kinases, as demonstrated by their inhibition with exogenous catalase that removes H2O2 or with diphenyleneiodonium (DPI) that inhibits NOX2. These results indicated that ERK and AKT are not essential of activation of NOX2 in AM and that reactive oxygen species can affect the signaling pathways leading to ERK and AKT activation. This is in agreement with studies in nonphagocytic cells, which showed regulation of these pathways by redox signaling. The mechanisms by which H2O2 affects ERK are still under investigation. We have examined the activation of upstream components of the ERK pathway, i.e. MEK, RAF, Ras, SHC in the presence of catalase or DPI. We have also used vanadate, an inhibitor of tyrosine phosphatase that can mimic the effects of H2O2 on ERK activation, suggesting that the target of H2O2 may be a tyrosine phosphatase acting on the ERK pathway.
SFRR 2004
S17-88 GASTROESOPHAGEAL REFLUX DISEASE (GERD) AS AN INFLAMMATORY DISEASE T. Yoshikawa,1 Y. Naito,1 and N. Yoshida1 1 Inflammation and Immunology, Kyoto Prefectural University of Medicine, Japan
We propose a hypothesis that an inflammatory response plays a crucial role in the pathogenesis of gastroesophageal reflux disease (GERD). Interleukin 8 (IL-8) has been shown to be proinflammatory, motogenic, mitogenic, and angiogenic and to play important roles in inflammation as well as tumor progression. Among the patients with GERD, there was a significant correlation between endoscopic esophagitis grading (LA classification) and the expression level of IL-8 mRNA determined by real-time PCR. In addition, the IL-8 mRNA expression was detected in patients suffering from heart burn without endoscopic abnormal findings. Epithelial cells, infiltrating neutrophils, and monocytes are candidates for IL-8 producing cells in the esophagus. Recently we have confirmed the IL-8 production from human esophageal epithelial cell line stimulated with tumor necrosis factor-a and interleukin 1h via activation of protein kinase C, protein tyrosine kinase, and p38 MAP kinase. Especially, bile acids in acidic condition markedly induce IL-8 expression in esophageal cells via activation of AP-1, NF-IL6, and NF-nB. Experimental rat models of reflux esophagitis have also demonstrated the marked expression of IL-8 like cytokines such as CINC-1 and CINC-2. In the present paper, we discuss the role of IL-8 associated inflammation in the course of GERD.
S17-89 DIESEL EXHAUST INDUCED PULMONARY INFLAMMATION: PROTECTIVE ROLE OF AIRWAY ANTIOXIDANTS F. J. Kelly, I. S. Mudway, S. T. Duggan, A. Blomberg, A. J. Frew, and T. Sandstro¨m School of Health and Life Sciences, King’s College, London, UK; University Hospital, Umea˚, Sweden; Southampton General Hospital, Southampton, UK
Inhalation of diesel exhaust (DE) can induce pulmonary inflammation and increased airway reactivity in healthy subjects possibly through the imposition of oxidative stress. To examine this, we exposed 25 healthy subjects to an environmentally relevant concentration of DE (PM10 100 Ag m 3, 0.6 ppm NO2 for 2-hours) and filtered air on separate occasions and monitored their antioxidant defences present at the air-lung interface. Subjects exposed to DE displayed a mild bronchconstriction and a trend toward neutrophilia in proximal airways. No depletion of ascorbate (AA), urate (UA) or reduced glutathione (GSH) was
seen at any level of the respiratory tract. Instead, an increased flux of GSH into the bronchial (p < 0.01) and nasal airways (p < 0.05) was observed. To establish whether the relatively mild inflammatory response was related to the movement of antioxidants into the airway, or simply reflected a lack of DE activity, we next examined the capacity of DE to deplete antioxidants from a synthetic model of the respiratory tract lining fluid. We found DE to have comparable oxidative activity to the transition metal rich residual oil fly ash (ROFA) particle, significantly depleting both AA and GSH. These data indicate that the antioxidant network present at the air-lung interface adapts rapidly to the oxidative burden placed upon it by exposure to DE, limiting acute inflammatory responses.
S17-90 CHRONIC INFLAMMATION IN END STAGE RENAL DISEASE: MARKERS OF OXIDATIVE STRESS AND REDOX MODIFICATION A. Iannone,1 S. Bergamini,1 E. Bellei,1 C. Rota,1 and A. Tomasi1 1 Department of Biomedical Sciences, University of Modena and Reggio Emilia, 41100 Modena, Italy
End-Stage Renal Disease (ESRD) is associated with an higher incidence cardiovascular disease due to accelerated atherosclerosis, and with clinical and/or laboratory signs of systemic inflammation. This condition has been considered to be related either, to the HD and to an increased prevalence of major cardiovascular risk factors. To test the presence of oxidative stress and redox modification in ESRD patients and to evaluate the role of HD, we performed assays in the blood at the beginning and at the end of the HD session. Patients have increased level of the inhibitor of the nitrogen monoxide (NO) synthase, the asymmetric dimethylarginine (ADMA), that could lead to a reduced NO-mediated vasodilation and contribute to the initiation and progression of atherosclerosis. The content of GSH in erythrocytes is decreased and there is the appearance in blood of malondialdehyde (MDA). The HD efficiently reduces the MDA levels and also plasma nitrites, which are high before dialysis, are reduced at the end of the HD session. During the dialysis most of the ascorbic acid and dehydroascorbic acid is removed, while there are not significant variation of atocopherol, h-carotene, lycopene and retinol.
SFRR 2004
S39
S17-91
S17-92
CHOLESTEROL OXIDATION PRODUCTS AND PRO-INFLAMMATORY EFFECTS
SIGNALING TO ERK BY THE NADPH OXIDASE OF ALVEOLAR MACROPHAGES
G. Leonarduzzi,1 A. Kadl,1 B. Sottero,1 P. Gamba,1 N. Leitinger,1 and G. Poli1 1
Department of Clinical and Biological Sciences, University of Torino, S. Luigi Gonzaga Hospital, 10043 Orbassano, Torino, Italy; Department of Vascular Biology and Thrombosis Research, University of Vienna, Austria
A considerable number of studies both in animals and humans have proved that prolonged high plasma cholesterol levels increase the risk of developing atherosclerosis but the mechanisms by which cholesterol contributes to initiation and progression of atherosclerotic lesions are still subjects of debate. Cholesterol oxidation products (oxysterols) are consistently recovered in atherosclerotic plaques and may markedly enhance chronic inflammation, fibrosis and programmed cell death. Of note, unoxidised cholesterol appears by far less reactive than oxysterols, pointing to oxidation as a crucial reaction to undergo for the sterol to exert proatherogenic effects. Support to these statements also derives from investigation we performed using U937 human promonocytic cells challenged with 7-ketocholesterol, unoxidized cholesterol or a biologically representative oxysterol mixture. By means of gene expression profiling, few genes encoding for chemokines, receptors, and adhesion molecules resulted to be over-expressed only when cells were treated with the oxysterol mixture. Such evidence was validated by real time RT-PCR and immunoenzymatic analyses. Experiments are now in progress to elucidate the intracellular pathways that oxysterols activate to exert inflammationrelated gene modulation.
S40
M. Torres1 and H. J. Forman2 1
The Saban Research Institute of Childrens Hospital Los Angeles, Department of Pediatrics, Keck School of Medicine, University of Southern California, Los Angeles, CA 90027, 2School of Natural Sciences, University of California, Merced, Merced, CA 95344, USA
Alveolar macrophages (AM) respond to various stimuli such as ADP or zymosan-activated serum (ZAS) with an increase in superoxide production due to the activation of the phagocyte NADPH oxidase, NOX2. The activation of NOX2 is multi-step process, which involves the activation of several kinases that phosphorylate components of NOX2. We previously showed that AM stimulation by ADP induces activation of NOX2 without activating ERK1/2, members of the mitogen-activated protein kinase family and AKT/PKB. Interestingly, ZAS did stimulate ERK and AKT but H2O2, the product of activation of NOX2 was necessary for activation of these kinases, as demonstrated by their inhibition with exogenous catalase that removes H2O2 or with diphenyleneiodonium (DPI) that inhibits NOX2. These results indicated that ERK and AKT are not essential of activation of NOX2 in AM and that reactive oxygen species can affect the signaling pathways leading to ERK and AKT activation. This is in agreement with studies in nonphagocytic cells, which showed regulation of these pathways by redox signaling. The mechanisms by which H2O2 affects ERK are still under investigation. We have examined the activation of upstream components of the ERK pathway, i.e. MEK, RAF, Ras, SHC in the presence of catalase or DPI. We have also used vanadate, an inhibitor of tyrosine phosphatase that can mimic the effects of H2O2 on ERK activation, suggesting that the target of H2O2 may be a tyrosine phosphatase acting on the ERK pathway.
SFRR 2004