Human monocytic U937 cells transfected with human hepatic inducible nitric oxide synthase exhibit leishmanicidal activity

In mice, the high inducible synthesis of nitric oxide (NO) resulting from inducible NO synthase (iNOS, NOS2) expression by macrophages (Mphi) is considered an essential component of the protective immune response against infection by intracellular pathogens. Conversely, in humans, the question of a role for NO as an antimicrobial defense mechanism has been the subject of much debate. Recently, however, iNOS expression by human Mphi and formation of NO or its derivatives have been reported both in vivo and in vitro, strongly suggesting that human Mphi are indeed capable of inducible NO synthesis. However, the conditions allowing NO production by human Mphi in culture remain poorly defined, rendering more difficult the study of the effector functions of NO in these cells. To alleviate this problem, cells of the U937 monocytoid line were engineered to express iNOS by transfection with human hepatic iNOS (DFGiNOS), leading to production of NO on supplementation with the cofactor tetrahydrobiopterin. We report that U937 cells, when differentiated with 1,25-dihydroxyvitamin D3 and retinoic acid, acquire a phenotype allowing infection by Leishmania parasites and maintain viable intracellular microorganisms up to 72 h post-infection. Leishmania survival in DFGiNOS cells is strongly decreased when the cells are treated with tetrahydrobiopterin. Intracellular killing is evident by 24 h and increases up to 72 h post-infection, and is inhibited by L-N5-(1-iminoethyl)ornithine, an inhibitor of NO synthesis. In contrast, superoxide anion does not appear to play a role in the killing of Leishmania by DGFiNOS U937 cells. The relevance of this model to the study of the mechanisms of intracellular killing by human macrophages is discussed.