Engineering of macrophages to produce IFN-γ in response to hypoxia

Activation of murine macrophages (M phi) requires the collaboration of signals derived from the immune system and the environment. In this study, we engineered a murine M phi cell line to become activated in response to an environmental signal, hypoxia, as the sole stimulus. Hypoxia is a condition of low oxygen tension, occurring in several pathological tissues, which acts in synergy with IFN-gamma to induce full M phi activation. We transfected the ANA-1 murine M phi cell line with a construct containing the IFN-gamma gene controlled by a synthetic promoter inducible by hypoxia (HRE3x-Tk), and we characterized the cellular and molecular biology of the engineered M phi under normoxia or hypoxia. Engineered M phi in normoxia expressed basal levels of IFN-gamma mRNA and protein that were strongly augmented by shifting the cells to hypoxia. Furthermore, they responded to the synthesized IFN-gamma with induction of IFN-responsive factor-1 and 2'-5'-oligoadenylate synthase expression. Under normoxic conditions, the engineered M phi had a significant constitutive level of la Ags and Fc receptors. Hypoxia induced further augmentation of Ia and Fe expression. Finally, hypoxia induced inducible NO synthase expression, and subsequent reoxygenation led to the production of NO. In conclusion, the engineered M phi, which produce IFN-gamma in an inducible manner, express new biochemical and functional properties in response to low oxygen environment as the sole stimulus, thereby circumventing the need for costimulation by other immune system-derived signals.