Mechanisms of synergistic cytokine-induced nitric oxide production in human alveolar epithelial cells

Nitric oxide (NO) derived from inducible NO synthase (iNOS) at sites of inflammation is closely related to host defense against infection and airway inflammation. Cytokines are known to stimulate NO production in human alveolar epithelial cells in a synergistic (nonlinear or nonadditive) manner. The mechanism of this synergy is not known. We measured the activation of the transcription factor NF-κB, the iNOS protein, and NO production in A549 monolayers (human alveolar epithelial cell line) in response to different combinations of IL-1β, INF-γ and TNF-α (100 ng/ml), and the cofactors FMN, FAD, and BH4. We found that both IL-1β and TNF-α could independently activate cytosolic NF-κB, direct its translocation into the nucleus, and induce iNOS monomer synthesis. In addition, different combinations of cytokines produced synergistic amounts of iNOS monomers. Exogenous BH4 (0.1 μM) had no impact on NO production induced by cytokine combinations that included IL-1β, but significantly enhanced NO production in the presence of INF-γ and TNF-α, and allowed TNF-α independently to produce NO. We conclude that there are at least three mechanisms of synergistic cytokine-induced NO production: (1) the biosynthesis of iNOS monomer due to nonlinear interactions by transcription factors, (2) synergistic cytosolic activation of NF-κB, and (3) parallel biosynthesis of BH4 in the presence of cytokine combinations that include IL-1β.