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

Activation of murine macrophages (M phi) requires the collaboration of sign als derived from the immune system and the environment. In this study, we e ngineered a murine M phi cell line to become activated in response to an en vironmental signal, hypoxia, as the sole stimulus. Hypoxia is a condition o f 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), an d we characterized the cellular and molecular biology of the engineered M p hi under normoxia or hypoxia. Engineered M phi in normoxia expressed basal levels of IFN-gamma mRNA and protein that were strongly augmented by shifti ng the cells to hypoxia. Furthermore, they responded to the synthesized IFN -gamma with induction of IFN-responsive factor-1 and 2'-5'-oligoadenylate s ynthase expression. Under normoxic conditions, the engineered M phi had a s ignificant constitutive level of la Ags and Fc receptors. Hypoxia induced f urther augmentation of Ia and Fe expression. Finally, hypoxia induced induc ible NO synthase expression, and subsequent reoxygenation led to the produc tion 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 circumvent ing the need for costimulation by other immune system-derived signals.