Effects of ibuprofen and hypoxia on neutrophil apoptosis in neonates

BACKGROUND: Ibuprofen is a cyclooxygenase inhibitor that is effective in treating patent ductus arteriosus in preterm infants. However, recent trials have suggested that it may increase the risk of developing necrotizing enterocolitis and chronic lung disease. Apoptosis of neutrophils is impaired in newborns, leading to reduced clearance of activated cells and possibly contributing to the susceptibility of infants to these inflammatory diseases. OBJECTIVES: In the present studies, we investigated the hypothesis that ibuprofen reduces neonatal neutrophil apoptosis in the setting of hypoxia or lipopolysaccharide (LPS). METHODS: Neutrophils and peripheral blood mononuclear cells were isolated from adult and cord blood and cultured in the presence or absence of ibuprofen (1.5 mM), hypoxia (<5% O2), and bacterial LPS (100 ng/ml). Apoptosis was quantified by measuring binding of FITC-Annexin V using flow cytometry. Cytokine concentrations in cell supernatants were measured by ELISA. RESULTS: After 24 h, 20% of adult and 14% of neonatal neutrophils were apoptotic. Apoptosis was reduced by hypoxia in both adult and neonatal cells. Ibuprofen further reduced neutrophil apoptosis, but only when the cells were cultured in the presence of mixed leukocytes. This suggests that the effects of ibuprofen on apoptosis are dependent on soluble products secreted by peripheral blood mononuclear cells. We found that production of tumor necrosis factor (TNF)-alpha by neonatal leukocytes was significantly increased by ibuprofen, and was further increased following exposure to ibuprofen in the presence of LPS and hypoxia. In contrast, production of macrophage inflammatory protein (MIP)-1alpha was not affected by treatment with ibuprofen, and ibuprofen blocked induction of this chemokine by LPS. CONCLUSION: We conclude that the net effect of ibuprofen on neutrophils is antiapoptotic, especially in the presence of hypoxia or LPS. This effect may be mediated, in part, by increased production of TNF-alpha by peripheral blood mononuclear cells. These data suggest that treatment of infants with ibuprofen, in the presence of infection and/or tissue hypoperfusion/hypoxia, may contribute to the development of inflammatory diseases.