title: Time-dependent apoptosis of alveolar macrophages from rats exposed to bleomycin: involvement of tnf receptor 2.
abstract: Tumor necrosis factor-alpha (TNF-a) is produced by alveolar macrophages (AM) in response to bleomycin (BLM) exposure. This cytokine has been linked to BLM-induced pulmonary inflammation, an early drug effect, and to lung fibrosis, the ultimate toxic effect of BLM. The present study was carried out to study the time dependence of apoptotic signaling pathways and the potential roles of TNF receptors in BLM-induced AM apoptosis. Male Sprague-Dawley rats were exposed to saline or BLM (1 mg/kg) by intratracheal instillation. At 1, 3, or 7 d postexposure, AM were isolated by bronchoalveolar (BAL) lavage and evaluated for apoptosis by ELISA. The release of cytochrome c from mitochrondria, the activation of caspase-3, -8, and -9, the cleavage of nuclear poly(ADP-ribose) polymerase (PARP), and the expression of TNF receptors (TNF-R1/p55 and TNF-R2/p75), TNF-R-associated factor 2 (TRAF2), and cellular inhibitor of apoptosis 1 (c-IAP1) were determined by immunoblotting. The results showed that BLM exposure induced AM apoptosis, with the highest apoptotic effect occurring at 1 d after exposure and gradually decreasing at 3 and 7 d postexposure, but still remaining significantly above the control level. The maximal translocation of cytochromec from mitochondria into the cytosol was observed at 1 d postexposure, whereas the activation of caspase-9 and caspase-3 and caspase-3-dependent cleavage of PARP was found to reach a peak level at 3 d postexposure. BLM exposure had no marked effect on AM expression of TNF-R1 or caspase-8 activation, but significantly increased the expression of TNF-R2 that was accompanied by a rise in c-IAP1 and a decrease in TRAF2. This induction of TNF-R2 by BLM was significant on d 1 and increased with greater exposure time. In vitro studies showed that pretreatment of naive AM with a TNF-R2 antibody significantly inhibited BLM-induced caspase-3 activity and apoptosis. These results suggest that BLM-induced apoptosis involves multiple pathways in a time-dependent manner. Since maximal BLM-induced AM apoptosis (1 d postexposure) preceded maximal changes in caspase-9 and -3 (3 d postexposure), it is possible that a caspase-independent mechanism is involved in this initial response. These results indicate that the sustained expression of TNF-R2 in AM by BLM exposure may sensitize these cells to TNF-a-mediated toxicity.
authors.affiliation: Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, West Virginia, USA.
authors.forename: H W
authors.initials: HW
authors.lastname: Zhao
authors.forename: S Y
authors.initials: SY
authors.lastname: Hu
authors.forename: M W
authors.initials: MW
authors.lastname: Barger
authors.forename: J K H
authors.initials: JK
authors.lastname: Ma
authors.forename: V
authors.initials: V
authors.lastname: Castranova
authors.forename: J Y C
authors.initials: JY
authors.lastname: Ma
mesh_terms: D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000633:toxicity*; D015703:Antigens, CD / Q000187:drug effects / Q000502:physiology; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D001761:Bleomycin / Q000633:toxicity*; D001992:Bronchoalveolar Lavage Fluid; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000187:drug effects; D045304:Cytochromes c / Q000187:drug effects; D004353:Drug Evaluation, Preclinical; D004781:Environmental Exposure / Q000009:adverse effects*; D004797:Enzyme-Linked Immunosorbent Assay; D015151:Immunoblotting; D007249:Inflammation; D007322:Instillation, Drug; D016676:Macrophages, Alveolar / Q000187:drug effects* / Q000502:physiology; D008297:Male; D011065:Poly(ADP-ribose) Polymerases / Q000187:drug effects; D011506:Proteins / Q000187:drug effects* / Q000502:physiology; D011658:Pulmonary Fibrosis / Q000139:chemically induced / Q000276:immunology / Q000473:pathology; D051381:Rats; D017207:Rats, Sprague-Dawley; D018124:Receptors, Tumor Necrosis Factor / Q000187:drug effects / Q000502:physiology; D047888:Receptors, Tumor Necrosis Factor, Type I; D015398:Signal Transduction / Q000187:drug effects; D047992:TNF Receptor-Associated Factor 2; D013997:Time Factors; D014178:Translocation, Genetic / Q000187:drug effects
chemical_list: D000903:Antibiotics, Antineoplastic; D015703:Antigens, CD; D011506:Proteins; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D047992:TNF Receptor-Associated Factor 2; D001761:Bleomycin; D045304:Cytochromes c; D011065:Poly(ADP-ribose) Polymerases; C505410:Casp3 protein, rat; C505528:Casp8 protein, rat; C506435:Casp9 protein, rat; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases