Reactive oxygen species may contribute to airway injury in patients with cystic fibrosis (CF) and iron catalyzes oxidant injury by promoting generation of highly reactive hydroxyl radicals. Iron in the lower respiratory tract may be free, ferritin bound (from which iron can be reductively mobilized), or transferrin bound (which generally prevents iron mobilization). Ferritin is composed of subunits that are heavy (H) or light (L), and H-rich ferritins have additional biologic effects including inhibition of lymphocyte proliferation and cell growth. To assess concentrations of iron and iron-binding proteins in the lower respiratory tract of patients with CF, we measured iron (ferrozine), L-ferritin, H-ferritin, and transferrin (enzyme-linked immunosorbent assay [ELISA]) in bronchoalveolar lavage (BAL) fluid recovered from stable patients with CF (n = 8), healthy nonsmokers (NS; n = 8), or heavy cigarette smokers (HS; n = 8). Iron was detected in BAL fluid from patients with CF and HS, but not NS, with higher iron concentrations in patients with CF (42.0 +/- 11.6 microgram/dl) than in HS (9.9 +/- 2.6 microgram/dl, p < 0.05). Ferritin was present in all BAL fluids, with higher total ferritin (L + H) in patients with CF (647 +/- 84 ng/ml) than in HS (181 +/- 25 ng/ml, p < 0.005) or NS (9 +/- 3 ng/ml, p < 0.0005). Ferritin recovered from HS and NS lungs was < 2% H type, whereas ferritin in CF lungs was > 40% H-type ferritin. Transferrin concentrations in BAL fluid were not different in any group. Tumor necrosis factor (TNF)-alpha was present only in BAL samples from patients with CF. To assess whether TNF-alpha contributed to H-ferritin accumulation in CF lungs, we treated lung epithelial cells (A549) with iron alone (FeSO(4), 10-40 microM) or with iron and TNF-alpha (5-20 ng/ml). Iron-treated A549 cells synthesized almost entirely L-ferritin whereas exposure to TNF-alpha with iron caused a dose-dependent increase in accumulation of H-type ferritin. These findings suggest that oxidant injury could be promoted in lungs of patients with cystic fibrosis by iron mobilized from extracellular ferritin and, in addition, that TNF-alpha-promoted accumulation of H-type ferritin may impair local immune function and cell growth.
Extracellular iron, which is predominantly bound by transferrin, is present in low concentrations within alveolar structures, and concentrations are increased in various pulmonary disorders. Iron accumulation by cells can promote oxidative injury. However, the synthesis of ferritin stimulated by metal exposure for intracellular iron storage is normally protective. The cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-1β may alter iron metabolism by alveolar cells. In this study, we assessed the effects of TNF-α and IL-1β on iron metabolism with a cell line with properties of type 2 alveolar epithelial cells (A549) exposed to non-transferrin-bound (NTBI; FeSO4) or transferrin-bound (TBI) iron. In addition, we assessed the cytotoxicity of these exposures by measuring the cell accumulation of malondialdehyde (MDA), a product of lipid peroxidation, and cell death (MTT assay and lactate dehydrogenase release). A549 cells treated with NTBI or TBI in concentrations up to 40 μM accumulated iron and synthesized predominantly L-type ferritin without accumulation of MDA or cell death. Treatment of A549 cells with TNF-α (20 ng) or IL-1β (20 ng) decreased cell transferrin-receptor expression and induced synthesis of H-type ferritin. TNF-α and IL-1β decreased the uptake of TBI; however, the uptake of NTBI was increased. Both cytokines enhanced total ferritin synthesis (H plus L types) in response to iron treatments due to enhanced synthesis of H-type ferritin. Coexposure to TNF-α and NTBI, but not to TBI, induced MDA accumulation and greater cytotoxicity (MTT and lactate dehydrogenase release) than TNF-α alone. These findings indicate that TNF-α and IL-1β modulate iron uptake by A549 cells, with differing effects on TBI and NTBI, as well as on H-ferritin synthesis. Enhanced iron uptake induced by TNF-α and NTBI was also associated with increased cytotoxicity to A549 cells.
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