- Email: [email protected]
Modulation of neutrophil activation by cigarette smoke
1141 II I
I1421 I
REGULATION OF APOPAIN /CASPASE 3 IN INFLAMMATORY HUMAN NEUTROPHILS: Jayasree Nath, Matthew Potenza, and
Ronald Wiggs. Department of Respiratory research, Walter Reed Army Institute of Research, Washington, DC. Although inflammatory neutrophils (PMNs) are known to undergo apoptosis, there is little information on the regulation of cellular events that modulate PMN apoptosis. The mammalian apopairUcaspase3, an ‘effector’ caspase, plays a pivotal role in apoptotic cell death via proteolytic cleavage of poly (ADP-ribose) polymerase. In our present in vitro study, induction of apopain activity was quantitated in freshly isolated human PMN-lysates, by using the fluorogenic peptide substrate Z-DEVD-AFC, in the absence and presence of a selective peptide inhibitor AC-DEVD-CMK. The enzyme activity was monitored in both unprimed and endotoxin (LPS)-primed PMNs. Effects of H,O,-mediated oxidative stress, SNAP, a stable NO donor, and ‘INFa, a pro-infbammatory cytokine, were assessed in both unstimulated and fmet-leu-phe (fMLF)stimulated PMNs. All assays were carried out at 37 C, for 3-4 brs. 500 pM H,O, induced a 2-3 fold increase in PMN apopain activity, which was abrogated by catalase. Interestingly, apopain activity in ‘INFIX- and LPS-primed PMNs were significantly decreased (3040%), as compared to unprimed PM&. Stimulation with fMLF did not cause any significant change in the activity of the enzyme. In contrast, incubation with 1e4 M SNAP caused a marked increase (>50%) in apopain activity, which was not observed when PMNs were stimulated with fMLF. The observed down-regulation of apopain/caspase3 in TNFa- and LPS-primed PMNs, and its upregulation in the presence of high concentrations of H,O, and NO, may be functionally relevant in the pathophysiology of acute inlhimmatory responses.
I
MODULATION OF NEUTROPHIL CIGARETTE SMOKE
ACTIVATION
BY
Abraham Reznrk Carroll Cross and Albert uan der Vlzet. Dept. UC Davis, CA 95616
-BM,
Cigarette smoking is a risk factor for respiratory and cardiovascular diseases, but its contributing actions are incompletely understood. Cigarette smokers commonly have higher levels of inflammatory cells and the multiple oxidants and nitrogen oxides present within cigarette smoke (CS) might interact with inflammatory components to induce of oxidative injury. To address this issue, we exposed isolated human neutrophils to gas-phase CS and analyzed the production of nitrating and chlorinating oxidants by trapping with hydroxyphenylacetate (HPA). We observed that CS is able to induce oxidation and nitration of HPA, but no chlorination. In contrast, stimulation of neutrophils with PMA caused oxidation and chlorination of this substrate, but no nitration. Stimulation of neutrophils in the presence of CS caused dramatically less HPA oxidation and chlorination, indicating that CS prevented activation of the NADPH oxidase or inhibited the activity of myeloperoxidase (MPO). The latter possibility was excluded based on unchanged peroxidase activity or unaltered oxidation and chlorination of HPA upon addition of HZ02 The apparent inactivation of NADPH oxidase was found to coincide with depletion of cellular GSH, suggesting modification of critical cysteine residues in one or more of the NADPH oxidase components. In support of this notion, GSH depletion and inhibition of NADPH oxidase activation was also observed after neutrophil exposure to comparable levels of acrolein, the major unsaturated aldehyde in CS. Additionally, protein adducts of acrolein or similar aldehydes could be detected after neutrophil exposure to either CS or acrolein. Diminished neutrophil activity in smokers may affect inflammatory/infectious conditions, and thereby contribute to
11431
) 144 1
MYELOPEROXIDASE-CATALYZED PROTEIN OXIDATION AND NITRATION AND EPITHELIAL TOXICITY IN CYSTIC FIBROSIS
REDOX-REGULATION OF IL-la mRNA STABILITY: AN EFFECT OF O2 AND HzOz ON mRNA TURNOVER Aparna C. Ranganathan, Ana M. Rodriguez, J.Andres Melendez, Department of Biochemistry and Molecular Biology, Albany Medical College, Albany, NY -12208
MAIN. NGUYW CARROLL CROSS, ALBERT VAN DER VLJET. Department of internalMedicine, University of California, Davis
Cystic fibrosis (CF)is a recessive autosomal disorder associated with increased vulnerability to bacterial lung infections. Active and chronic inflammation in the lungs of CF patients is marked by the production of various oxidants as a result of the activity of heme peroxidases such as myeloperoxidase (MPO). MPO catalyzes the oxidation of various substrates, such as a halide anion or nitrite, and thereby induces the oxidation and nitration of amino acid residues, which at high levels can cause cellular toxicity. CF sputum was found to contain elevated levels of MPO and oxidative products characteristic of MPO activity. Since pulmonary function is inversely correlated with peroxidase concentrations, this suggests that MPO-derived oxidant reactions contribute significantly to cytotoxicity in CF-affected lungs. Studies with cultured epithelial cells confirm that MPO in CF sputum can contribute to oxidant-mediated cell damage, though protein oxidation occurred predominantly in sputum proteins rather than in cellular proteins. CF sputum-exposed cells, after being secondarily exposed to oxidant systems, revealed enhanced levels of nitrotyrosine relative to cells not exposed to CF sputum. This suggests an accelerated protein modification within the respiratory tract of CF patients possibly induced by elevated levels of MI’O. Increased generation of MPO-catalyzed oxidizing and nitrating species within the respiratory tract of CF subjects chronically may result in epithelial toxicity and dysfunction. This reveals a potential route of damage in the pathology of cystic fibrosis.
s54
OXYGEN
The regulation of IL-la is of fundamental importance. ILla mRNA contains in its Buntranslated region (UTR’s), adenylate-uridylate rich elements (ARES) which are RNA destabilizing elements. Our laboratory has previously shown that MnSOD regulates ILI- a levels post-transcriptionally, by altering mRNA stability. To determine the influence of changes in cellular oxygen levels on the 2.4kEl IL-la transcript, deletion constructs of IL-la were transiently transfected into HT-1080 fibrosarcoma cells in either 21% or 3% 02. Northern blot analysis indicates that constructs containing 500-1000 bps of the coding region are highly expressed in 21% 02 but down regulated in 3% 02. To define the MnSOD dependent regulation of IL-la, analysis of IL-la levels was performed in cells which co-express both MnSOD and catalase. These studies indicate that dramatic overexpression of MnSOD increases IL-la levels, while the overexpression of both prevents this increase. This effect was more pronounced when catalase was overexpressed in the mitochondria. This suggests that H202, a product of the dismutation reaction, regulate IL -la levels.
’ 9
9
I1421 I
REGULATION OF APOPAIN /CASPASE 3 IN INFLAMMATORY HUMAN NEUTROPHILS: Jayasree Nath, Matthew Potenza, and
Ronald Wiggs. Department of Respiratory research, Walter Reed Army Institute of Research, Washington, DC. Although inflammatory neutrophils (PMNs) are known to undergo apoptosis, there is little information on the regulation of cellular events that modulate PMN apoptosis. The mammalian apopairUcaspase3, an ‘effector’ caspase, plays a pivotal role in apoptotic cell death via proteolytic cleavage of poly (ADP-ribose) polymerase. In our present in vitro study, induction of apopain activity was quantitated in freshly isolated human PMN-lysates, by using the fluorogenic peptide substrate Z-DEVD-AFC, in the absence and presence of a selective peptide inhibitor AC-DEVD-CMK. The enzyme activity was monitored in both unprimed and endotoxin (LPS)-primed PMNs. Effects of H,O,-mediated oxidative stress, SNAP, a stable NO donor, and ‘INFa, a pro-infbammatory cytokine, were assessed in both unstimulated and fmet-leu-phe (fMLF)stimulated PMNs. All assays were carried out at 37 C, for 3-4 brs. 500 pM H,O, induced a 2-3 fold increase in PMN apopain activity, which was abrogated by catalase. Interestingly, apopain activity in ‘INFIX- and LPS-primed PMNs were significantly decreased (3040%), as compared to unprimed PM&. Stimulation with fMLF did not cause any significant change in the activity of the enzyme. In contrast, incubation with 1e4 M SNAP caused a marked increase (>50%) in apopain activity, which was not observed when PMNs were stimulated with fMLF. The observed down-regulation of apopain/caspase3 in TNFa- and LPS-primed PMNs, and its upregulation in the presence of high concentrations of H,O, and NO, may be functionally relevant in the pathophysiology of acute inlhimmatory responses.
I
MODULATION OF NEUTROPHIL CIGARETTE SMOKE
ACTIVATION
BY
Abraham Reznrk Carroll Cross and Albert uan der Vlzet. Dept. UC Davis, CA 95616
-BM,
Cigarette smoking is a risk factor for respiratory and cardiovascular diseases, but its contributing actions are incompletely understood. Cigarette smokers commonly have higher levels of inflammatory cells and the multiple oxidants and nitrogen oxides present within cigarette smoke (CS) might interact with inflammatory components to induce of oxidative injury. To address this issue, we exposed isolated human neutrophils to gas-phase CS and analyzed the production of nitrating and chlorinating oxidants by trapping with hydroxyphenylacetate (HPA). We observed that CS is able to induce oxidation and nitration of HPA, but no chlorination. In contrast, stimulation of neutrophils with PMA caused oxidation and chlorination of this substrate, but no nitration. Stimulation of neutrophils in the presence of CS caused dramatically less HPA oxidation and chlorination, indicating that CS prevented activation of the NADPH oxidase or inhibited the activity of myeloperoxidase (MPO). The latter possibility was excluded based on unchanged peroxidase activity or unaltered oxidation and chlorination of HPA upon addition of HZ02 The apparent inactivation of NADPH oxidase was found to coincide with depletion of cellular GSH, suggesting modification of critical cysteine residues in one or more of the NADPH oxidase components. In support of this notion, GSH depletion and inhibition of NADPH oxidase activation was also observed after neutrophil exposure to comparable levels of acrolein, the major unsaturated aldehyde in CS. Additionally, protein adducts of acrolein or similar aldehydes could be detected after neutrophil exposure to either CS or acrolein. Diminished neutrophil activity in smokers may affect inflammatory/infectious conditions, and thereby contribute to
11431
) 144 1
MYELOPEROXIDASE-CATALYZED PROTEIN OXIDATION AND NITRATION AND EPITHELIAL TOXICITY IN CYSTIC FIBROSIS
REDOX-REGULATION OF IL-la mRNA STABILITY: AN EFFECT OF O2 AND HzOz ON mRNA TURNOVER Aparna C. Ranganathan, Ana M. Rodriguez, J.Andres Melendez, Department of Biochemistry and Molecular Biology, Albany Medical College, Albany, NY -12208
MAIN. NGUYW CARROLL CROSS, ALBERT VAN DER VLJET. Department of internalMedicine, University of California, Davis
Cystic fibrosis (CF)is a recessive autosomal disorder associated with increased vulnerability to bacterial lung infections. Active and chronic inflammation in the lungs of CF patients is marked by the production of various oxidants as a result of the activity of heme peroxidases such as myeloperoxidase (MPO). MPO catalyzes the oxidation of various substrates, such as a halide anion or nitrite, and thereby induces the oxidation and nitration of amino acid residues, which at high levels can cause cellular toxicity. CF sputum was found to contain elevated levels of MPO and oxidative products characteristic of MPO activity. Since pulmonary function is inversely correlated with peroxidase concentrations, this suggests that MPO-derived oxidant reactions contribute significantly to cytotoxicity in CF-affected lungs. Studies with cultured epithelial cells confirm that MPO in CF sputum can contribute to oxidant-mediated cell damage, though protein oxidation occurred predominantly in sputum proteins rather than in cellular proteins. CF sputum-exposed cells, after being secondarily exposed to oxidant systems, revealed enhanced levels of nitrotyrosine relative to cells not exposed to CF sputum. This suggests an accelerated protein modification within the respiratory tract of CF patients possibly induced by elevated levels of MI’O. Increased generation of MPO-catalyzed oxidizing and nitrating species within the respiratory tract of CF subjects chronically may result in epithelial toxicity and dysfunction. This reveals a potential route of damage in the pathology of cystic fibrosis.
s54
OXYGEN
The regulation of IL-la is of fundamental importance. ILla mRNA contains in its Buntranslated region (UTR’s), adenylate-uridylate rich elements (ARES) which are RNA destabilizing elements. Our laboratory has previously shown that MnSOD regulates ILI- a levels post-transcriptionally, by altering mRNA stability. To determine the influence of changes in cellular oxygen levels on the 2.4kEl IL-la transcript, deletion constructs of IL-la were transiently transfected into HT-1080 fibrosarcoma cells in either 21% or 3% 02. Northern blot analysis indicates that constructs containing 500-1000 bps of the coding region are highly expressed in 21% 02 but down regulated in 3% 02. To define the MnSOD dependent regulation of IL-la, analysis of IL-la levels was performed in cells which co-express both MnSOD and catalase. These studies indicate that dramatic overexpression of MnSOD increases IL-la levels, while the overexpression of both prevents this increase. This effect was more pronounced when catalase was overexpressed in the mitochondria. This suggests that H202, a product of the dismutation reaction, regulate IL -la levels.
’ 9
9