Activation of alveolar macrophages (AM) in the production of tumor necrosis factor (TNF)-alphaby bleomycin (BLM) is linked to AM apoptosis and the development of pulmonary fibrosis. AM may contribute to their own death through expression of TNF receptor (TNFR) 1 and 2. The present study was carried out to characterize the role of the TNFR in the mechanism(s) of BLM-induced apoptosis in AM. Sprague Dawley rats were instilled intratracheally with saline or BLM at 1 mg/kg body weight. At 1, 3, or 7 days post-exposure, AM were isolated by bronchoalveolar lavage, and apoptosis was determined by ELISA. The activation of caspases 3, 8, and 9, the release of cytochrome c from mitochrondria, the cleavage of nuclear poly(ADP-ribose) polymerase (PARP), and the amount of TNFR1 and TNFR2 in AM were monitored by immunoblotting. The results showed that BLM treatment significantly induced AM apoptosis at all exposure time points, with peak apoptosis occurring at 1 day post BLM exposure. BLM treatment, at 1, 3 or 7 days post exposure, significantly increased active caspase 3 level with enhanced caspase 3 activity and increased PARP cleavage in AM in comparison to the control. The maximum activation of caspase 3 and PARP fragmentation occurred in AM from rats exposed to BLM for 3 days. The amount of cytochrome c released into cytosol was gradually increased with time after BLM treatment and peaked on day 7. This coincides with expression of TNFR2, which was significantly induced in BLM-exposed AM with a peak level at 7-day post exposure. In contrast, BLM treatment did not affect AM expression of TNFR1. BLM exposure also significantly activated caspase 9 but not caspase 8 in AM. These results show that BLM-induced apoptosis is TNFR1/caspase-8 independent, but involves over-expression of TNFR2, the release of cytochrome c from mitochondria, activation of caspases 9 and 3, and the cleavage of the DNA repair protein PARP.
The Toxicologist. Society of Toxicology 42nd Annual Meeting and ToxExpo, Cutting-Edge Science, Networking, New Perspectives, March 9-13, 2003, Salt Lake City, Utah