All these inflammatory mediators together play a crucial role in the orchestration of an inflammatory response, particularly in neutrophil recruitment, representing a different type of effector RO4929097 nmr cells. Neutrophil sequestration and migration into alveoli remain pathohistological hallmarks of ARDS, with neutrophils being key effector cells, which further destruct lung tissue . The process of programmed cell death, or apoptosis, is known to play a major regulatory role in maintaining many biological processes, not least of which is the inflammatory response, such as in ALI/ARDS. Two major apoptosis pathways
in mammalian cells are known so far: (i) the intrinsic or mitochondrial pathway with involvement of Bcl-2 at the outer membrane of mitochondria, cytochrome c release and activation of caspase-9; and (ii) extrinsic or death receptor pathway with activation of caspase-8 upon binding of death activator to Fas- and tumour necrosis factor (TNF)-receptor at the surface of the cell. Both pathways converge at the level of caspase-3 activation . Apoptosis results in destruction of proteins by caspases as well as in fragmentation of the DNA. Finally, apoptotic cells are eliminated by phagocytes.
Inappropriate activation or inhibition of apoptosis can lead to disease either because ‘undesired’ cells develop prolonged survival or because ‘desired’ cells die prematurely . The purpose of this study was to evaluate in vitro apoptosis rate and pathway of effector and target cells at different time-points upon injury with endotoxin and hypoxia, both factors 3-mercaptopyruvate sulfurtransferase which selleck kinase inhibitor might contribute to ALI in vivo. We were interested to
assess if upon injury different cell types undergo apoptosis in a similar way. Our hypothesis was that within the group of effector or target cells, the cells would experience the same kind of apoptosis. Specific pathogen-free male Wistar rats (250–300 g) were purchased from Janvier (Le Genest-St Isle, France). Rats were anaesthetized with subcutaneously administered Narketan (ketamine 10%, Kepro, Utrecht, the Netherlands) 0·8–1 ml/kg and Rompun (Xylazin 2%, Streuli Pharma, Uznach, Switzerland) 0·25–0·5 ml/kg. All animal experiments and animal care were approved by the Swiss Veterinary Health Authorities. Alveolar macrophages (CRL-2192; American Type Culture Collection, Rockville, MD, USA) were established from normal Sprague–Dawley rat alveolar macrophage cells obtained by lung lavage, cloned and subcloned three times. The cells exhibit characteristics of macrophages and are sensitive to endotoxin. Cells from passages not higher than 5 were used. Cells were cultured in nutrient mixture F-12 Ham (Ham’s F-12; Invitrogen Corporation, Carlsbad, CA, USA), completed with 15% fetal bovine serum (FBS), 5% penicillin/streptomycin (10 000 U/l) and 5% HEPES. Overnight, before starting the experiments, cells were incubated with Ham’s F-12 with 1% FBS.