Hyperoxia-induced neutrophil infux in neonatal rats may contribute to impaired lung development through oxidative DNA damage. To determine whether blocking neutrophil influx prevents DNA damage, we treated newborn rats with 95% O2 beginning at birth, and at 3 and 4 d with nonimmune immunoglobulin G (IgG) (control) or anti-cytokine-induced neutrophil chemoattractant (CINC). At 8 d, lungs were inflation-fixed. Random sections were labeled using terminal transferase nick end-labeling (TUNEL), and DNA oxidation was measured using anti-8-OH-2'-deoxyguanosine (OHdG). To determine whether hyperoxia-induced TUNEL represented apoptosis, we labeled sections with anti-Bax (proapoptotic) and anti-Bcl-2 (antiapoptotic). We labled additional sections with anti-M30, directed against an epitope formed by caspase 6 digestion of cytokeratin 18 during apoptosis. Hyperoxia induced marked increases in TUNEL and OHdG signal in lung parenchymal cells, which was substantially prevented by treatment with anti-CINC. The large effects of hyperoxia on TUNEL were not accompanied by substantial effects on Bax, Bcl-2, or M30. We conclude that neutrophil influx during hyperoxia damages DNA by nicking and oxidation, and that blocking neutrophil influx can prevent this. Effects of 95% O2 on TUNEL are not primarily due to apoptosis in this model. Neutrophil-mediated oxidative DNA damage may contribute to abnormal lung development in newborns subjected to significant oxidative stress.

Hyperoxia may contribute to lung disease in newborns through effects on alveolar neutrophils which predominate in respiratory distress syndrome and other acute lung injuries. Neutrophil chemokines such as interleukin-8 (IL-8) regulate chemoattraction, and are elevated in tracheal aspirates of newborns who develop bronchopulmonary dysplasia (BPD). Blockade of neutrophil chemokines may reduce hyperoxia-induced inflammatory lung injury and BPD. We therefore tested the hypothesis that hyperoxia contributes to elevations of rat neutrophil chemokines, cytokine-induced neutrophil chemoattractant-1 (CINC-1), and macrophage inflammatory protein-2 (MIP-2) in newborn rat lung. Newborn rats were exposed to air or 95% O(2) for 8 d. CINC-1 and MIP-2 were measured in whole lung homogenates by ELISA. Newborn 95% O(2)-exposed animals were given anti-CINC-1 or anti-MIP-2, 1, 5, or 10 microg on Days 3 and 4 of 95% O(2) exposure. Bronchoalveolar lavage (BAL) was performed after perfusion on day 6 to evaluate airway neutrophils, and myeloperoxidase (MPO) was measured in perfused whole lung. Lungs were examined histologically and immunohistochemically for effects of 95% O(2) +/- antichemokine. CINC-1 and MIP-2 increased nearly tenfold by Day 8 95% O(2) treatment versus air control. CINC-1 and MIP-2 immunolabeling was increased in alveolar macrophages and alveolar epithelium in 95% O(2). Anti-CINC-1 and anti-MIP-2 treatment at every dose reduced neutrophil number > 90% in BAL. Anti-CINC-1 10 microg reduced tissue MPO by 50%. Antichemokine treatment on days 3 and 4 prevented alveolar septal thickening and reduced chemokine immunolabeling on Day 6. Hyperoxia-induced neutrophil influx is mediated in part by CINC-1 and MIP-2 in newborn rats and can be partially prevented by treatment with anti-CINC-1 and anti-MIP-2.