Expression of the inducible NO synthase in human monocytic U937 cells allows high output nitric oxide production

Nitric oxide (NO) produced by inducible NO synthase (iNOS, NOS-2) is an imp ortant component of the macrophage-mediated immune defense toward numerous pathogens. Murine macrophages produce NO after cytokine activation, whereas , under similar conditions, human macrophages produce low levels or no NO a t all, Although human macrophages can express iNOS mRNA and protein on acti vation, whether they possess the complete machinery necessary for NO synthe sis remains controversial, To define the conditions necessary for human mon ocytes/macrophages to synthesize NO when expressing a functional iNOS, the human monocytic U937 cell line was engineered to synthesize this enzyme, fo llowing infection with a retroviral expression vector containing human hepa tic iNOS (DFGiNOS). Northern blot and Western blot analysis confirmed the e xpression of iNOS in transfected U937 cells both at the RNA and protein lev els, NOS enzymatic activity was demonstrated in cell lysates by the convers ion of L-[H-3]arginine into L-[H-3]citrulline and the production of NO by i ntact cells was measured by nitrite and nitrate accumulation in culture sup ernatants, When expressing functional iNOS, U937 cells were capable of rele asing high levels of NO, NO production was strictly dependent on supplement ation of the culture medium with tetrahydrobiopterin (BH4) and was not modi fied by stimulation of the cells with different cytokines, These observatio ns suggest that (I) human monocytic U937 cells contain all the cofactors ne cessary for NO synthesis, except BRL and (2) the failure to detect NO in cy tokine-stimulated untransfected U937 cells is not due to the presence of a NO-scavenging molecule within these cells nor to the destabilization of iNO S protein. DFGiNOS U937 cells represent a valuable human model to study the role of NO in immunity toward tumors and pathogens.