cytochrome-c-t and Cystic-Fibrosis

Pulmonary infections with Pseudomonas aeruginosa are a serious clinical problem and are often lethal. Because many strains of P. aeruginosa are resistant to antibiotics, therapeutic options are limited. Neutrophils play an important role in the host's early acute defense against pulmonary P. aeruginosa. Therefore, it is important to define the mechanisms by which P. aeruginosa interacts with host cells, particularly neutrophils.. Here, we report that pyocyanin, a membrane-permeable pigment and toxin released by P. aeruginosa, induces the death of wild-type neutrophils; its interaction with the mitochondrial respiratory chain results in the release of reactive oxygen species (ROS), the activation of mitochondrial acid sphingomyelinase, the formation of mitochondrial ceramide, and the release of cytochrome c from mitochondria. A genetic deficiency in acid sphingomyelinase prevents both the activation of this pathway and pyocyanin-induced neutrophil death. This reduced death, on the other hand, is associated with an increase in the release of interleukin-8 from pyocyanin-activated acid sphingomyelinase-deficient neutrophils but not from wild-type cells.. These studies identified the mechanisms by which pyocyanin induces the release of mitochondrial ROS and by which ROS induce neutrophil death via mitochondrial acid sphingomyelinase.. These findings demonstrate a novel mechanism of pyocyanin-induced death of neutrophils and show how this apoptosis balances innate immune reactions.

Topics: Animals; Cell Death; Cell Line; Ceramides; Cystic Fibrosis; Cytochromes c; HL-60 Cells; Humans; Interleukin-8; Jurkat Cells; Liver; Membrane Potential, Mitochondrial; Mice, Inbred C57BL; Mitochondria; Neutrophils; Pseudomonas aeruginosa; Pseudomonas Infections; Pyocyanine; Rats; Reactive Oxygen Species; Sphingomyelin Phosphodiesterase

In the absence and in the resolution of inflammatory responses, neutrophils rapidly undergo spontaneous apoptosis. Here we report about a new apoptosis pathway in these cells that requires calpain-1 activation and is essential for the enzymatic activation of the critical effector caspase-3. Decreased levels of calpastatin, a highly specific intrinsic inhibitor of calpain, resulted in activation of calpain-1, but not calpain-2, in neutrophils undergoing apoptosis, a process that was blocked by a specific calpain-1 inhibitor or by intracellular delivery of a calpastatin peptide. Further support for the importance of the calpastatin-calpain system was obtained by analyzing neutrophils from patients with cystic fibrosis that exhibited delayed apoptosis, associated with markedly increased calpastatin and decreased calpain-1 protein levels compared with neutrophils from control individuals. Additional studies were designed to place calpain-1 into the hierarchy of biochemical events leading to neutrophil apoptosis. Pharmacological calpain inhibition during spontaneous and Fas receptor-induced neutrophil apoptosis prevented cleavage of Bax into an 18-kDa fragment unable to interact with Bcl-xL. Moreover, calpain blocking prevented the mitochondrial release of cytochrome c and Smac, which was indispensable for caspase-3 processing and enzymatic activation, both in the presence and absence of agonistic anti-Fas receptor antibodies. Taken together, calpastatin and calpain-1 represent critical proximal elements in a cascade of pro-apoptotic events leading to Bax, mitochondria, and caspase-3 activation, and their altered expression appears to influence the life span of neutrophils under pathologic conditions.

Topics: Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; bcl-X Protein; Calcium-Binding Proteins; Calpain; Carrier Proteins; Caspase 3; Caspases; Cell Death; Cells, Cultured; Cystic Fibrosis; Cytochromes c; Densitometry; Dose-Response Relationship, Drug; Enzyme Activation; fas Receptor; Humans; Immunoblotting; Intracellular Signaling Peptides and Proteins; Microscopy, Confocal; Mitochondrial Proteins; Models, Biological; Models, Molecular; Neutrophils; Precipitin Tests; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Subcellular Fractions; Time Factors

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine known to activate macrophages and T cells. In this study, we demonstrate that recombinant MIF delays apoptosis of neutrophils in vitro. MIF action is dose and time dependent as well as specific since it was abolished with a neutralizing anti-MIF antibody. MIF, like G-CSF, delayed cleavage of the proapoptotic members of the Bcl-2 family Bid and Bax in neutrophils, suggesting that MIF inhibits apoptosis pathways proximal to mitochondria activation. Indeed, MIF also prevented release of cytochrome c and Smac from the mitochondria and subsequent activation of the critical effector caspase-3 in these cells. Moreover, we observed increased MIF plasma levels in patients with cystic fibrosis, a heterogeneous recessive genetic disorder associated with bacterial infections and delayed neutrophil apoptosis. In conclusion, MIF is a survival cytokine for human neutrophils, a finding with potential pathologic relevance in infectious diseases.

Topics: Apoptosis; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Carrier Proteins; Caspase 3; Caspases; Cells, Cultured; Complement Membrane Attack Complex; Complement System Proteins; Cystic Fibrosis; Cytochromes c; Glycoproteins; HeLa Cells; Humans; Kinetics; Macrophage Migration-Inhibitory Factors; Mitochondria; Neutrophils; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Signal Transduction