Pteridines and cytokines induce apoptotic cell death in alveolar epithelial cells
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KeywordsNitric Oxide Apoptotic Cell Death Alveolar Epithelial Cell Neopterin Reactive Oxygen Intermediate
The pteridines neopterin and 7.8-dihydroneopterin influence the cellular oxidant-antioxidant balance as well as the expression of the inducible nitric oxide synthase (iNOS) gene. Since apoptotic cell death can he initiated by reactive oxygen intermediates and nitric oxide (NO) we studied whether both pterdines induce apoptotic cell death in vitro.
Materials and methods
As cell culture model we selected the rat alveolar epithelial cell line L2. Confluent L2 cells were incubated for 24 h with neopterin (1 μM-1000 μM). 7.8-dihydroneopterin (1 μM-1000 μM), the cytomix (interferon-γ IFN-γ, 100 U/ml plus tumor necrosis factor-α, TNF-α 500 U/ml) as well as the combination of the pteridines and the cytomix. Apoptosis was estimated by FACS analysis. iNOS gene expression was investigated after 9 h incubations by an RT-PCR. Synthesis of the stable NO-metabolites nitrite and nitrate was determined in the cell-free supernatants incubated for 24 h by a modified Griess reaction.
Neopterin as well as 7.8-dihydroncopterin induced a significant increase of percentual apoptotic cells. Maximal apoptosis was found with 100 γM neopterin (mean ± SEM = 22.7 ± 2.5%; n = 6) and 100 μM 7.8-dihydroneopterin (mean ± SEM = 21.9 ± 2.5%: n = 6). respectively. Co-incubation of both pteridines with the cytomix lead to a significant higher apoptosis than the cytomix alone. In contrast to the cytomix. no iNOS gene expression and no NO release could be detected after incubation with neopterin as well as 7,8-dihydroneopterin.
We conclude that neopterin and 7,8-dihydroneopterin are per se inducers of apoptosis. They enhance apoptotic cell death by IFN-γ and TNF-α. In contrast to these cytokines apoptotic cell death due to the pteridines is not mediated by nitric oxide. The pronounced apoptosis by the pteridines may be of importance in inflammatory pulmonary diseases associated with an activation of the cellular immune system.